Lipoplexes formed from sugar-based gemini surfactants undergo a lamellar-to-micellar phase transition at acidic pH. Evidence for a non-inverted membrane-destabilizing hexagonal phase of lipoplexes.

The present study aims at a better understanding of the mechanism of transfection mediated by two sugar-based gemini surfactants GS1 and GS2. Previously, these gemini surfactants have been shown to be efficient gene vectors for transfection both in vitro and in vivo. Here, using Nile Red, a solvatochromic fluorescent probe, we investigated the phase behavior of these gemini surfactants in complexes with plasmid DNA, so-called lipoplexes. We found that these lipoplexes undergo a lamellar-to-non-inverted micellar phase transition upon decreasing the pH from neutral to mildly acidic. This normal (non-inverted) phase at acidic pH is confirmed by the colloidal stability of the lipoplexes as shown by turbidity measurements. We therefore propose a normal hexagonal phase, H(I), for the gemini surfactant lipoplexes at acidic endosomal pH. Thus, we suggest that besides an inverted hexagonal (H(II)) phase as reported for several transfection-potent cationic lipid systems, another type of non-inverted non-bilayer structure, different from H(II), may destabilize the endosomal membrane, necessary for cytosolic DNA delivery and ultimately, cellular transfection.

pH-dependent phase behavior of carbohydrate-based gemini surfactants. The effects of carbohydrate stereochemistry, head group hydrophilicity, and nature of the spacer.

The pH-dependent phase behavior and hydroxide-ion adsorption ability of a series of (reduced) carbohydrate-based gemini surfactants were studied between pH 2 and 12. Static and dynamic light scattering were employed to address transitions in the aggregate morphologies and cryo-electron microscopy was used to provide further evidence for the morphologies present in solution. Changes in aggregate structure as a result of a change in solution pH and an accompanying change in protonation state or a change in molecular structure can be rationalized in terms of the variations in the packing parameter. In this paper we have focused our attention on the size of the carbohydrate moiety, the carbohydrate stereochemistry and the nature of the spacer (hydrophobic vs hydrophilic). At near neutral pH, most of the gemini surfactants form vesicles. Upon lowering of the pH, the vesicles undergo a transition toward wormlike micelles followed by a transition to spherical micelles. Upon increasing the solution pH, flocculation occurs due to charge neutralization followed at still higher pH by redispersion and charge reversal of the vesicles through the specific adsorption of hydroxide ions to the vesicular surface. Upon decreasing head group size at constant, but low, degrees of protonation, the packing parameter has a tendency to become larger than one resulting in the formation of inverted phases. Upon further decrease in the head group size, oil droplets are observed. In case of a hydrophobic spacer, the carbohydrate stereochemistry affects the pH of the transitions, but not the type of the transitions. By contrast, for a hydrophilic spacer, the pH of the transitions remains unaffected. Adsorption of hydroxide ions at basic pH follows similar trends, but was only found for vesicles and oil droplets. The large range of structural variations that we have examined allows a better understanding of the requirements for the phase transitions for carbohydrate-based gemini surfactants as well as for the physisorption of hydroxide ions to interfaces in general.

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.

pH-dependent phase behavior of carbohydrate-based gemini surfactants. Effect of the length of the hydrophobic spacer.

The phase behavior of a series of carbohydrate-based gemini surfactants with varying spacer lengths was studied using static and dynamic light scattering between pH 2 and 12. Cryo-electron microscopy pictures provide evidence for the different morphologies present in solution. The spacer length of the gemini surfactants was varied from two to 12 methylene units. At near neutral pH, spherical vesicles were obtained for gemini surfactants with a spacer shorter than 10 methylene units, whereas nonspherical vesicles were obtained for spacer lengths of 10 and 12. Upon decreasing the pH, the vesicles underwent transitions toward worm-like micelles and spherical micelles for a spacer length of six and larger, whereas for shorter spacers, these transitions are not observed. For the shortest spacer at low pH, perforated vesicles are observed, and vesicles built from the gemini surfactant with a spacer of four methylene units only underwent a transition toward worm-like micelles. Upon increasing the pH to slightly basic values, flocculation followed by redispersion upon charge reversal was observed up to a spacer length of eight methylene units. The redispersal is explained by hydroxide-ion binding to the uncharged vesicular surface. By contrast, vesicles formed from the gemini surfactants with 10 and 12 methylene units only undergo a transition toward inverted phases. The observations can be understood in terms of the packing parameter.

Characterization and transfection properties of lipoplexes stabilized with novel exchangeable polyethylene glycol-lipid conjugates.

The positive charge of cationic-lipid/DNA complexes (lipoplexes) renders them highly susceptible to interactions with the biological milieu, leading to aggregation and destabilization, and rapid clearance from the blood circulation. In this study we synthesized and characterized a set of novel amphiphiles, based on N-methyl-4-alkylpyridinium chlorides (SAINTs), to which a PEG moiety is coupled. Plasmids were fully protected in lipoplexes prepared from cationic SAINT-2 lipid and stabilized with SAINT-PEGs. Our results demonstrate that SAINT-PEG stabilization is transient, and permits DNA to be released from these lipoplexes. The rate of SAINT-PEG transfer from lipoplexes to acceptor liposomes was determined by the nature of the lipid anchor. Increased hydrophobicity, by lengthening the alkyl chain, resulted in a decrease of the rate of DNA release from the lipoplexes. Chain unsaturation had the opposite effect. Similarly, the in vitro transfection potency of lipoplexes containing PEG-SAINT derivatives was sensitive to the length and (un)saturation of the alkyl chain. However, the internalization of SAINT-PEG stabilized lipoplexes is determined by their charge, rather than by the concentration of the polymer conjugate. Lipoplexes targeted to cell-surface epithelial glycoprotein 2, by means of a covalently coupled monoclonal antibody, were specifically internalized by cells expressing this antigen.

Interference of serum with lipoplex-cell interaction: modulation of intracellular processing.

We have investigated the mechanism of lipoplex-mediated transfection, employing a dialkyl pyridinium surfactant (SAINT-2), and using serum as a modulator of complex stability and processing. Particle size and stability determine lipoplex internalization, the kinetics of intracellular processing, and transfection efficiency. Clustered SAINT-2 lipoplexes are obtained in the absence of serum (-FBS lipoplexes), but not in its presence (+FBS lipoplexes), or when serum was present during lipoplex formation [FBS], conditions that mimic potential penetration of serum proteins. The topology of DNA in [FBS] lipoplexes shifts from a supercoiled, as in -FBS lipoplexes, to a predominantly open-circular conformation, and is more prone to digestion by DNase. Consistently, atomic force microscopy revealed complexes with tubular extensions, reflecting DNA that protrudes from the lipoplex surface. Interestingly, the internalization of [FBS] lipoplexes is approximately three-fold higher than that of -FBS and +FBS lipoplexes, yet their transfection efficiency is approximately five-fold lower. Moreover, in contrast to -FBS and +FBS complexes, [FBS] complexes were rapidly processed into the late endosomal/lysosomal degradation pathway. Intriguingly, transfection by [FBS] complexes is greatly improved by osmotic rupture of endocytic compartments. Our data imply that constraints in size and morphology govern the complex' ability to interact with and perturb cellular membranes, required for gene release. By extrapolation, we propose that serum may regulate these parameters in an amphiphile-dependent manner, by complex 'penetration' and modulation of DNA conformation.

Gemini surfactants: new synthetic vectors for gene transfection.

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.

Retro-Diels-Alder Reaction in Aqueous Solution: Toward a Better Understanding of Organic Reactivity in Water.

The retro-Diels-Alder (RDA) reaction of anthracenedione 1a proceeds considerably faster in aqueous solutions than in organic solvents. Addition of organic solvents to water retards the reaction, whereas glucose induces a modest acceleration. SDS micelles induce a considerable retardation, but even at high concentrations of surfactant (complete micelle-substrate binding), the cycloreversion is not fully inhibited. Correlation with data for solvatochromic indicators strongly suggest that the origin of the water-induced acceleration involves primarily enhanced hydrogen bonding of water to the activated complex for the RDA reaction of 1a. Activation parameters support this view. A comparison of the present results with previous kinetic data for bimolecular and intramolecular Diels-Alder reactions provides insights into the contributions of hydrogen-bond and hydrophobic interactions to the aqueous accelerations of the latter two types of reactions.

Solvent Effects on a Diels-Alder Reaction Involving a Cationic Diene: Consequences of the Absence of Hydrogen-Bond Interactions for Accelerations in Aqueous Media.

In order to study the influence of hydrogen-bond interactions on the accelerations of Diels-Alder reactions in water and highly aqueous mixed solvent systems, second-order rate constants for the Diels-Alder reaction of acridizinium bromide (1a) with cyclopentadiene (CP) have been measured in aqueous media and organic solvents. Only modest rate accelerations were found in water-rich media. This is attributed to the absence of hydrogen-bonding groups in the reactants. Comparison with cycloadditions of CP with 9-carbomethoxyacridizinium bromide (1b), acrylonitrile (3), and methyl vinyl ketone (4), which do contain hydrogen-bond acceptors, reveals substantially larger aqueous accelerations. These results demonstrate that hydrogen bonding is a major factor in aqueous accelerations. Also rate constants for the cycloaddition of CP to 1a in surfactant solutions were determined. Micellar catalysis is observed in SDS solutions, due to binding of both the diene and the dienophile to the anionic micellar surface.

Aggregation behavior of p-n-alkylbenzamidinium chloride surfactants.

The aggregation behavior of a novel class of surfactants, p-n-alkylbenzamidinium chlorides, has been investigated. The thermodynamics of aggregation of p-n-decylbenzamidinium chloride mixed with cationic and anionic cosurfactants has been studied using isothermal titration calorimetry. For mixtures of p-n-decylbenzamidinium chloride with n-alkyltrimethylammonium chlorides, the aggregation process is enthalpically more favorable than for the pure n-alkyltrimethylammonium chlorides, probably caused by diminished headgroup repulsion due to charge delocalization in the amidinium headgroup. A critical aggregation concentration between 3 and 4 mM has been extrapolated for p-n-decylbenzamidinium chloride at 40 degrees C, around two times lower than that of similar surfactants without charge delocalization in the headgroup and well comparable to that of similar surfactants with charge delocalization in the headgroup. In mixtures of p-n-decylbenzamidinium chloride with either sodium n-alkylsulfates or sodium dodecylbenzenesulfonate, evidence is found for the formation of bilayer aggregates by the pseudo-double-tailed catanionic surfactants composed of p-n-decylbenzamidinium and the anionic surfactant. These aggregates are solubilized to mixed micelles by excess free anionic surfactant at the measured critical aggregation concentration.

Reversible optical control of monolayers on water through photoswitchable lipids.

We have obtained molecular insights into a monolayer of azobenzene-based photoswitchable lipids self-assembled on water, using the surface sensitive technique vibrational sum-frequency generation spectroscopy in combination with surface pressure measurements. The photolipids can undergo wavelength-dependent, light-triggered cis/trans and trans/cis isomerization, allowing for reversible control of the surface pressure and the molecular ordering of the lipids in the monolayer. If the photoswitchable lipid is embedded in a layer with conventional phospholipids, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), we show that the surface pressure and molecular ordering of DPPC can be influenced by switching the azobenzene-based lipid between its two states. Remarkably, the state with the higher surface pressure (cis-state) is characterized by a lower degree of molecular order. This counterintuitive result can be understood by noting that the azobenzene moiety in the cis state has a higher dipole moment and therefore favors interaction with water. The surface free energy of the system is lowered (increase of surface pressure) by electrostatic interactions with the lipid headgroups at the interface, resulting in a loop formation of the lipid tail with the cis-azobenzene. This disorder in the tail of the photoswitchable lipid perturbs as well the ordering of DPPC.

Photochemically induced disturbance of the alkyl chain packing in vesicular membranes.

In previous reports, we presented the synthesis and properties of double-tailed azobenzene-substituted phosphate amphiphiles (Kuiper et al. Synthesis 2003, 695 and Kuiper et al. Langmuir 2004, 20, 1152). We also reported that an ion channel can be regulated by trans-cis isomerization of these amphiphiles, which were incorporated in the membrane (Folgering et al. Langmuir 2004, 20, 6985). In the present study, the effect of trans-cis isomerization of both single- and double-tailed azobenzene-substituted amphiphiles on the aggregation and packing behavior has been studied. The phase transition temperature of a membrane and the thermal half-life times of the cis azobenzene-substituted amphiphiles in membranes have been measured. Furthermore, the synthesis and properties of single-tailed azobenzene-substituted phosphate amphiphiles are described and compared with those of the double-tailed analogues. The single-tailed azobenzene-substituted phosphates have a low solubility in water and form micelles, sheets, and crystals. In all cases the trans-cis isomerization leads to a disturbance of the chain packing. Both single- and double-tailed cis azobenzene-substituted phosphates lowered the main phase transition temperature of bilayer membranes. The effect increased when the azobenzene moiety was situated closer to the head group. Accordingly, the half-life times of the cis azobenzene group was shorter when the azobenzene group was positioned closer to the head group for both the single- and double-tailed amphiphiles. Interestingly, the thermal cis-trans isomerization of the single-tailed azobenzene-substituted phosphates was faster in a DOPC membrane than that for the free monomer in aqueous solution.

Gene delivery by cationic lipid vectors: overcoming cellular barriers.

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex-cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection.

Chain-length and solvent dependent morphological changes in sodium soap fibers.

Sodium soap fibers with varying alkyl chain lengths were studied by cryotransmission electron microscopy and differential scanning calorimetery in water and water-propylene glycol mixtures. The morphology of the lamellar fibers was found to be dependent on the chain length of the alkyl chain and the solvent polarity. Cryoelectron microscopy revealed that short-chain (C10-C14) sodium soaps have the bilayer plane perpendicular to the fiber width, which enables one to see the bilayer striations on the fibers, whereas long-chain (C16-C20) sodium soaps have bilayer planes parallel to the fiber width, and the bilayer striations are not visible. This change in morphology is accompanied by a change in dissolution enthalpy.

Hydroxide-ion binding to nonionic interfaces in aqueous solution.

The mechanism of hydroxide ion binding to nonionic surfaces is explored by variation of the properties of the water-aggregate interface and by variation of the type of the aggregate.

The influence of phenyl and phenoxy modification in the hydrophobic tails of di-n-alkyl phosphate amphiphiles on aggregate morphology.

A series of di-n-alkyl phosphate amphiphiles containing phenyl and phenoxy groups in the hydrophobic tails were synthesised, and their aggregation behaviour was investigated using fluorescence spectroscopy, differential scanning calorimetry, and cryo-electron microscopy. The aggregates displayed a wide variety of aggregate morphologies. The incorporation of a phenyl group into the end or in the middle of the alkyl chain lowered the main phase transition temperature, resulting in closed vesicles only above the phase transition temperature. Introducing a phenoxy group at the end of the alkyl chain resulted in open bilayer structures and bicelles.

pH-Dependent aggregation properties of mixtures of sugar-based gemini surfactants with phospholipids and single-tailed surfactants.

Sugar-based gemini surfactants (GSs) display rich pH-dependent phase diagrams and are considered to be promising candidates as gene- and drug-delivery vehicles for biomedical applications. Several sugar-based GSs form vesicles around neutral pH. The vesicular dispersions undergo transitions toward wormlike micelles and spherical micelles at acidic pH, whereas flocculation followed by redispersion upon charge reversal is observed at basic pH. The influence of various amounts of the double-tailed phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and of the single-tailed surfactants lyso-PC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) and OTAC (octadecyltrimethylammonium chloride) on the phase behavior of GS1 (1,8-bis(N-octadec-9-yl-1-deoxy-D-glucitol-1-ylamino)3,6-dioxaoctane) was determined as a function of pH, in water and in water at physiological ionic strength. The pH corresponding to the phase transitions and the characteristics of the aggregates were determined by means of a combination of physical techniques: static and dynamic light scattering (SLS and DLS), fluorescence spectroscopy, cryo-TEM and diffusion- and (31)P NMR. The results show that the additives affect the phase behavior of the GS1 dispersions in a pH-dependent fashion. In the presence of double-tailed phospholipids, a higher degree of protonation of GS1 must be reached to observe micelle formation, whereas single-tailed surfactants affect these transitions only slightly. In the presence of increasing amounts of lyso-PC, the pH range of flocculation becomes more narrow, indicating the increased hydration of the vesicles. The pH of redispersion after charge reversal is particularly sensitive to the presence of positively charged additives. It is suggested that the cationic headgroups disturb the hydrogen-bond structure of water at the vesicular surface, hampering OH(-) binding. The effect of an increase in ionic strength to physiological values is found to be modest, except for the dispersions containing the positively charged additives.

Physisorption of hydroxide ions from aqueous solution to a hydrophobic surface.

We present results from detailed molecular dynamics simulations revealing a counterintuitive spontaneous physical adsorption of hydroxide ions at a water/hydrophobic interface. The driving force for the migration of the hydroxide ions from the aqueous phase is the preferential orientation of the water molecules in the first two water layers away from the hydrophobic surface. This ordering of the water molecules generates an electrical potential gradient that strongly and favorably interacts with the dipole moment of the hydroxide ion. These findings offer a physical mechanism that explains intriguing experimental reports indicating that the interface between water and a nonionic surface is negatively charged.

Vesicular catalysis of an S(N)2 reaction: toward understanding the influence of glycolipids on reactions proceeding at the interface of biological membranes.

The kinetics of the S(N)2 reaction of a series of aromatic alkylsulfonates with water and bromide ions in membrane mimetic media have been investigated. These media include vesicles formed from only synthetic amphiphiles, vesicles composed only of phospholipids and mixtures of these components. Special focus is placed on the influence of the addition of n-dodecyl-beta-glucoside as a mimic for glycolipids. The kinetic data have been analyzed by using the pseudophase model for bimolecular reactions. Contrary to previous results on a base-catalyzed E2 reaction (Org. Biomol. Chem. 2004, 2, 1789-1799), the presence of n-dodecyl-beta-glucoside at the vesicular surface does not lead to large rate accelerations for the S(N)2 reaction. In fact, when present at 50 mol % (i.e., the additive covers 34% of the vesicular surface) these glycolipid mimics appear not to affect the bimolecular rate constants, but they only decrease the local water concentration by about 40%. The reactivity of water at the surface of vesicles that are formed from cationic amphiphiles appears to be increased about 10-fold relative to the reactivity of water in the bulk liquid, whereas in zwitterionic vesicles the reactivity is comparable to that in bulk water. The obtained rate constants are also compared to micellar rate constants.