Sofia Candeloro De Sanctis

Tailoring Lipoplex Composition to the Lipid Composition of Plasma Membrane: A Trojan Horse for Cell Entry?

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.

Effect of cholesterol on the formation and hydration behavior of solid-supported niosomal membranes.

The effect of cholesterol on the formation and hydration behavior of solid-supported polysorbate 20 (Tween 20)/cholesterol self-assemblies was investigated by means of in situ energy-dispersive X-ray diffraction in a wide range of relative humidity (0.4 < RH < 1). At low hydration, Tween 20 and cholesterol were found to demix, with the latter molecules forming crystallites with a pseudobilayer structure (d approximately = 34 A). Water adsorption promoted the progressive solubilization of cholesterol crystallites. When in the presence of enough cholesterol, the formation of niosomal bilayer membranes rich in Tween 20 occurred (RH approximately = 0.985). Upon further hydration, two distinct regimes associated with remarkable changes in the niosomal membrane structure were identified. In the first regime (0.985 < RH < 0.988), the swelling of the lamellar d spacing was due to the enlargement of the membrane thickness. In the second regime, the structure of Tween 20/cholesterol membranes was quite insensitive to hydration, and the thickness of the intermembrane water layer increased substantially. Remarkably, the curve of the calculated number of waters per surfactant molecule showed a distinct break at RH approximately 0.988, suggesting that the observed structural change in niosomal membranes was most likely due to the completion of the filling of the Tween 20 hydration shell. At full hydration, niosomal membranes exhibited the same lamellar d spacing of niosomes vesicles in aqueous solution. The process completely reversed upon dehydration.

Toward the rational design of lipid gene vectors: Shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of lipoplexes and the efficiency of DNA release

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.

From crystal to micelle: A new approach to the micellar structure

The structure of sodium and rubidium deoxycholate micellar aggregates in aqueous solutions was found to be helical and to be stabilized mainly by polar interactions. Astonishingly, the lateral surface of the helix is covered by nonpolar groups and the interior part is filled with cations surrounded by water molecules, as in the case of an inverted micelle. This helical model was inferred from the crystal structures of sodium and rubidium deoxycholates and proved by spectroscopic and diffractometric experimental data. The strategy of the approach to the determination of the micellar structure and the comparison with another model, previously proposed for the bile salt micelles, are reported. On the basis of some results obtained for sodium tauro- and glyco-deoxycholates, micellar models are suggested which could account for the biological function of these important conjugated bile salts.

Effect of membrane charge density on the protein corona of cationic liposomes: Interplay between cationic charge and surface area

Here we investigate the effect of membrane charge density on the protein corona that forms upon exposure of cationic liposomes to human plasma. We show that the protein corona around cationic liposomes is not uniquely defined by membrane charge density, but it is deeply affected by both the overall cationic charge and the available surface area. Remarkably, changes in protein composition are largely removed when a correcting quantity accounting for the ratio between the cationic charge and the surface area of different cationic liposome formulations is considered.

Spontaneous formation of pigmentary precipitates in bile salt-depleted rat bile and its prevention by micelle-forming bile salts.

During studies on the effect of bile salt-pool depletion in the bile-fistula rat (adult male Sprague-Dawley), the spontaneous formation of an orange-brown precipitate was noted. The nature of this phenomenon and its relationship to BS and calcium concentration was investigated in depth. Bile from 18 animals was collected in the dark into transparent tubes containing sodium azide, ascorbic acid, and glucaro-1,4-lactone. The tubes were flushed with nitrogen, sealed, and incubated at 37 degrees C. The pigmentary precipitate formed in all the bile salt-depleted (less than 3-5 mM) bile samples (i.e., those collected after 5-7 h of external biliary drainage), but not in bile salt-rich biles. It appeared within 30-240 min after collection, both in bile samples collected at room temperature and at 37 degrees C, initially as a pale flocculation and then slowly sedimenting to form, after centrifugation, a solid, dark-orange pellet. There were no pH changes during incubation, and bile cultures were negative. Under polarizing microscopy, the precipitate appeared amorphous, and there was no evidence of birefringence. High-performance liquid chromatography showed that unconjugated bilirubin was the prevalent pigmentary component, but significant amounts of monoconjugated bilirubin also coprecipitated. Lipid chemistry showed the presence of lecithin (80.1% of total lipids), which was rich in palmitoyl and linoleoyl fatty acids, and of fatty acids (predominantly palmitic and oleic). Infrared spectroscopy and x-ray diffraction showed the presence of calcium bilirubinate and palmitate. In-vivo replenishment of the bile salt pool by intravenous infusion of either taurocholate or taurochenodeoxycholate (1 mumol/min) completely prevented the pigmentary precipitation. In vitro experiments showed inhibition of the precipitate formation by the addition of individual bile salt in concentrations approximating their critical micellar concentration. Precipitate formation was hastened by the addition of calcium chloride (4-12 mM), but only in bile salt-depleted biles. As the composition of the precipitate closely resembles that of human brown-pigment stones and sludge, these findings may provide new insights into an understanding of the pathogenesis of pigment gallstone disease.

Crystal structures of bile salts: Sodium taurocholate

Crystals of sodium taurocholate (NaC26H44NO7S · 2.5 H2O) belonging to the triclinic space groupP1 have unit cell parametersa = 12.731 (2),b = 16.104 (2),c = 7.628 (1) ⫗A, α =83.40 (1),β = 101.20 (1), γ = 105.35 (1)°, and two molecules in the asymmetric unit. The refinement, carried out on 4424 observed reflections, gaveR = 0.059 andRw = 0.066. The packing is characterized by bilayers, formed by antiparallel monolayers and with nonpolar outermost surfaces, held together by van der Waals interactions. Inside the bilayers there are channels, lined with polar groups, and filled by sodium ions and water molecules. A structural unit has been identified that could provide a reasonable model for the micellar aggregates of this bile salt.

A possible helical model for sodium glycocholate micellar aggregates

Sodium glycocholate crystallizes in the tetragonal space group14 witha =b = 27.793(4),c = 7.937(1) Å andZ = 8. Refinement based on 2290 observed reflections led to a conventionalR = 0.10. The bile salt molecules are arranged in a helix with 21 symmetry stabilized mainly by polar interactions. Four helices are held together by hydrogen bonds involving water molecules, giving rise to hydrophilic channels, with a small cross section, which can include water molecules. The packing of these tetramers form hydrophobic channels containing some disordered acetone and water molecules. The helices will be checked as a model for the micellar aggregates of this important conjugated bile salt, following the same strategy successfully applied to sodium deoxycholate.

Potential energy calculations on the 2∶1 inclusion compound between deoxycholic acid and (+)-camphor

The crystal structure of the 2∶1 inclusion compound between deoxycholic acid and (+)-camphor has been solved by other authors. We have studied its crystal packing by potential energy calculations. Four arrangements of camphor, satisfactory from an energy point of view, were found and used to refine the crystal structure by a least-squares procedure. The results seem to indicate that more than one orientation is possible for the guest molecule and that the X-ray data do not allow us to establish which guest-molecule arrangements are actually present.