The interdigitated gel phase in mixtures of cationic and zwitterionic phospholipids.

To examine the phase behavior of mixtures of zwitterionic and cationic lipids we used three derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). All three lipids are uniquely capable of spontaneously forming the interdigitated gel phase (LßI) under typical hydration conditions. The P-O-ethyl derivative, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC), was chosen as the cationic lipid. For the zwitterionic lipids, we use the ether-linked 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine, (DHPC) and the fluorine substituted 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine (F-DPPC). Differential scanning calorimetry (DSC) and fluorescence spectroscopy were used to analyze the lipid mixtures. The F-DPPC/EDPPC mixtures are interdigitated at all lipid ratios below the main transition temperature (Tm). In addition, EDPPC stabilizes the interdigitated gel phase of DHPC until the ripple gel phase (Pß') is eliminated and only the LßI to liquid crystalline phase (Lα) main transition remains. These results demonstrate that mixtures of cationic and zwitterionic lipids can be compatible with the interdigitated phase.

Effects of cis- and trans-unsaturated lipids on an interdigitated membrane.

The effects of adding cis- and trans-unsaturated lipid to a fully interdigitated membrane were examined using differential scanning calorimetry (DSC) and X-ray diffraction. A monofluorinated analog of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used as the interdigitated lipid. The single fluorine atom on the end of the sn-2 chain allows 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC) to spontaneously form the interdigitated gel phase (LßI) below the main transition temperature (Tm). The cis 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and equivalent trans lipid 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) are strongly disfavored to form the LßI phase. Wide-angle X-ray scattering (WAXS) data demonstrate that the unsaturated lipids progressively disrupt the intermolecular packing at higher concentrations. Small-angle X-ray scattering (SAXS) data show that as the ratio of unsaturated lipid increases, the amount of interdigitated lipid decreases. The cis isomer is more disruptive and inhibits interdigitation more effectively than the trans isomer.

Influence of the interdigitated gel phase in mixtures of ether-linked and monofluorinated ester-linked phospholipids.

To evaluate the influence of the hydrocarbon chain linkage on the thermodynamic phase behavior of spontaneously interdigitating lipids, mixtures of ether-linked 1,2-di-O-hexadecyl-phosphocholine (DHPC) and ester-linked 1-palmitoyl-2-[16-fluoropalmitoyl]sn-glycero-3-phosphocholine (F-DPPC) were studied. A combination of differential scanning calorimetry (DSC), fluorescence spectroscopy, and transmittance spectrophotometry was used. Small amounts of F-DPPC increase the pretransition temperature (T(p)) between the interdigitated gel phase (L(ß)I) and the ripple gel phase (P(ß)'). There are some signs of immiscibility where the non-interdigitated lipid is present in the phase diagram. However, at around 20mol.% F-DPPC, the pretransition merges with the main transition and is no longer detectable as a separate entity. Additionally, the T(m) hysteresis increases steadily with higher mole fractions of F-DPPC. These results support that incorporating F-DPPC progressively stabilizes the L(ß)I phase of DHPC until the membrane is fully interdigitated below the main transition temperature (T(m)). The mixtures of F-DPPC and DHPC are miscible once the membrane is entirely interdigitated in the gel phase. Therefore, the ability of both lipids to interdigitate is an important factor controlling gel phase miscibility. Our results also demonstrate that the gel phase behavior of DHPC is highly sensitive to changes in its environment.

Effects of cholesterol on phospholipid membranes: inhibition of the interdigitated gel phase of F-DPPC and F-DPPC/DPPC.

Unlike the parent phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the monofluorinated analog, 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC), spontaneously forms an interdigitated gel phase (L(ß)I) below the main transition temperature (T(m)). We have examined the effects of introducing cholesterol to F-DPPC and 1:1 F-DPPC/DPPC membranes using a combination of DSC, optical density, fluorescence intensity and polarization, (31)P NMR, and X-ray diffraction techniques. Cholesterol increases the fluidity of the gel phase, broadens the main transition, and decreases the main transition enthalpy. However, these results also reveal that there is an unusually large degree of phase coexistence between the L(ß)I and non-interdigitated gel phases when cholesterol is added. Cholesterol encourages this phase segregation by partitioning into the thicker non-interdigitated domains. At higher cholesterol concentrations, the majority or all of the L(ß)I phase of F-DPPC and 1:1 F-DPPC/DPPC is eliminated and is replaced by a non-interdigitated liquid-ordered (l(o)) phase with properties similar to DPPC/cholesterol. Consequently, cholesterol mitigates the influence the CF moiety has on the thermodynamic phase behavior of F-DPPC. Our findings demonstrate that there are multiple characteristics of cholesterol-rich membranes that disfavor interdigitation.

Effects of pentanol isomers on the phase behavior of phospholipid bilayer membranes.

Differential scanning calorimetry (DSC) was used to analyze the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of pentanol isomers. The concentration of each pentanol isomer needed to induce the interdigitated phase was determined by the appearance of a biphasic effect in the main transition temperatures, the onset of a hysteresis associated with the main transition from the gel-to-liquid crystalline phase, and the disappearance of the pretransition. Lower threshold concentrations were found to correlate with isomers of greater alkyl chain length while branching of the alkyl chain was found to increase biphasic behavior. The addition of a methyl group to butanol systems drastically decreased threshold concentrations. However, as demonstrated in the DPPC/neopentanol system, branching of the alkyl chain away from the -OH group lowers the threshold concentration while maintaining a biphasic effect.

Properties of phosphatidylcholine in the presence of its monofluorinated analogue.

In aqueous solution, the monofluorinated phospholipid 1-palmitoyl-2-[16-fluoropalmitoyl]sn-glycero-3-phosphocholine (F-DPPC) interdigitates without the use of inducing agents. To understand the thermal and physical properties of this unique lipid, F-DPPC was combined with the non-fluorinated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC). Differential scanning calorimetry (DSC) was used to determine the miscibility and thermotropic phase behavior of these binary lipid mixtures. In addition, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and a DPH-labeled analogue of DPPC, 2-(3-(diphenylhexatrienyl) propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (beta-DPH HPC, aka DPH-PC or DPHpPC), were used to detect interdigitation. In F-DPPC, the fluorescence intensity of both probes decreased a similar amount and to a degree that is consistent with an interdigitated system. We also determined that there are two separate effects of increasing the ratio of F-DPPC in the DPPC/F-DPPC system. With low amounts of F-DPPC, there is little evidence that the system is heavily interdigitated. Instead, we hypothesize that the introduction of F-DPPC provides nucleation sites that alter the kinetics, reversibility, and temperature of the main transition (T(m)). At higher mol% of F-DPPC, we propose that interdigitated F-DPPC-rich domains form to create a phase-segregated system. While DPPC/F-DPPC was highly miscible, the DAPC/F-DPPC system was significantly less miscible. Additionally, we observed that DAPC/F-DPPC samples have reduced solubility in water, which affected the acquisition of fluorescence data. However, our DSC results indicate the existence of DAPC-rich and F-DPPC-rich components. Furthermore, this data support that the mixing was disruptive to lipid packing and that the presence of DAPC hinders the interdigitation of F-DPPC.