Threshold concentrations for some alcohol-induced interdigitation.
DSC is a versatile technique and has been used for decades to study hydrated phospholipid membranes [1-4]. It can even be used to analyze whole cell samples . For pure lipids, DSC can accurately determine the phase transition temperatures and the associated enthalpies. As a consequence, how the chemical structure of lipids translates into thermodynamic properties can be systematically studied. In addition to determining the physical properties of pure lipids, the miscibility and phase behavior of lipid mixtures can be determined.
The detailed review of the interdigitated phase written by Slater and Huang in 1988 provides an excellent outline of the properties of the interdigitated phase and the relevant analytical techniques . Furthermore, the meticulous studies of Koynova and Caffrey describe how systematic changes in lipid chemistry can affect their phase behavior [7-9]. Lipids with asymmetrical acyl chains that form either mixed- or partially-interdigitated phases have also been thoroughly investigated [7,10-12]. This review focuses on the interdigitated phase of fully hydrated phospholipids with hydrocarbon chain lengths of equal size. We pay special attention to recently discovered interdigitated systems and the chemicals that can induce or inhibit lipid interdigitation.
For simplicity, we have centered our review around the extensively studied lipid, 1,2-dipalmitoyl-
|DPPC/alcohol||Threshold concentration (M)||References|
|Methanol||2.75 ± 0.35|||
|Ethanol||1.10 ± 0.10|||
|1-propanol||0.39 ± 0.03|||
|2-propanol||0.52 ± 0.03|||
|1-butanol||0.16 ± 0.02|||
|Isobutanol||0.17 ± 0.02|||
|0.22 ± 0.02|||
|0.27 ± 0.02|||
|1-pentanol||0.07 ± 0.01|||
|2-pentanol||0.10 ± 0.01|||
|3-pentanol||0.11 ± 0.01|||
|3-methyl-2-butanol||0.10 ± 0.01|||
|2-methyl-1-butanol||0.08 ± 0.01|||
|3-methyl-1-butanol||0.08 ± 0.01|||
|2-methyl-2-butanol||0.13 ± 0.01|||
|neopentanol||0.08 ± 0.01|||
There are multiple types of interdigitation (Figure 4). The type of interdigitation that forms is heavily dependent on the structure and symmetry of the hydrocarbon chains . Interdigitated lipid systems can further be separated into two classes: spontaneous and induced. We use “spontaneous” to describe lipids that self-assemble into the interdigitated gel phase when fully hydrated under typical preparation procedures and at ambient pressure. Some notable recent examples are highlighted, such as cationic lipids and lipids with monofluorinated acyl chains. Whether or not a particular lipid spontaneously interdigitates is determined by the balance of properties that favor and disfavor interdigitation. Lipids often have conflicting characteristics regarding the ability to form the interdigitated phase. Consequently, there is no simple formula for determining which lipids will spontaneously interdigitate without relying on experimental data.
As can be seen in Figure 4, the structural difference between the interdigitated and non-interdigitated phases can be substantial. In the non-interdigitated membrane, both ends of the hydrocarbon chains meet in the membrane midplane (Figure 4A). Two well-defined leaflets are formed and there is a thick hydrophobic core. In the fully-interdigitated membrane, the thickness of the interdigitated phase is greatly reduced and there is the loss of the membrane midplane. There is an increase in the spacing between the polar lipid head groups and the ends of the lipid hydrocarbon chains become more exposed to the aqueous interface . The difference is most dramatic in the fully interdigitated phase compared to the non-interdigitated membrane (Figure 4A and 4B). In the partially-interdigitated system, the longer chain extends to the other side of the membrane and aligns with the apposing shorter chain (Figure 4C). In the mixed-interdigitated membrane, the short hydrocarbon chains line up with each other and the full-length chain extends to the other side of the membrane (Figure 4D). Lyso lipids also form a fully interdigitated structure (Figure 4E) .
2.1. Thermodynamics of phosphatidylcholine membranes
PCs are common in mammalian membranes and have well known phase transitions . The most ubiquitous of these is the gel-to-liquid crystalline transition, often referred to as the melting or main transition. This transition is relatively rapid and is highly reversible . It is characterized by the co-operative melting of the hydrocarbon chains and a high enthalpy DSC peak . The liquid crystalline (
2.2. Chemically-induced interdigitation of phosphatidylcholines
The most widely studied chemical inducer of interdigitation is ethanol. In non-interdigitated phospholipid membranes, ethanol tends to adsorb to the head groups, especially the region near the hydrocarbon chains [38,39]. In particular, the carbonyl groups of the glycerol backbone of phospholipids are thought to be the favored hydrogen bonding sites for ethanol . Ethanol displaces water when it adsorbs to the head group, which increases the head group volume and decreases the order of the hydrocarbon chains [13,41,42]. The increase in head group volume leads to increased chain tilting and creates energetically unfavorable voids in the hydrocarbon region of non-interdigitated membranes, encouraging the creation of the
There are three main characteristics of the chemically-induced interdigitated phase in the DSC thermograms of saturated PCs: the presence of biphasic phase behavior, an increase in
The “biphasic effect” indicates two independent interactions within different concentration ranges [46,47]. The biphasic effect is most strongly characterized by an initial decrease in the
The shape and magnitude of the
Increasing the alcohol content well above the threshold concentration lowers the
The biphasic behavior is also reflected in the increase in the main transition enthalpy as the alcohol concentration increases [15,16]. Often, the rate of change in the transition enthalpy above and below the threshold concentration is different (Figure 6). This effect also depends on the alcohol chain length and isomer used. For instance, this difference is clear with
A property that accompanies the biphasic effect is the emergence of hysteresis in the main transition [15,16,48,49]. The hysteresis as it relates to DSC is defined as the difference in the transition temperature between heating and cooling scans. This corresponds to the reversibility and kinetics of the transition. The return to the interdigitated phase with a decrease in temperature is a slow process and is therefore less reversible [48,51]. For systems that do not interdigitate, such as phosphatidylethanolamine (PE) lipids, the addition of alcohol does not affect the transition hysteresis .
The disappearance of the pre-transition is another consistent property of alcohol-induced interdigitation of saturated PCs. The decrease in the
By comparing the threshold concentrations of different chemicals, they can be ranked on their effectiveness at inducing the interdigitated phase. For instance, the threshold concentrations for alcohol-induced interdigitation systematically decreases as the lipid hydrocarbon chain length increases (Table 1). The isomers with the most solubility in water are the least effective at inducing interdigitation, as shown by the increase in threshold concentrations . Additionally, the more soluble an isomer is in water, the less effectively it depresses both the temperature of the pre-transition and the main transition prior to the threshold concentration .
The ether-linked analogue of DPPC, 1,2-di-
2.3. Pressure-induced interdigitation
It is well established that the application of hydrostatic pressure favors interdigitation in a multitude of lipid systems (Tables 2 and 3). As hydrostatic pressure is applied, the intermolecular distance between adjacent lipids is reduced and molecular packing becomes denser . By changing the packing structure of the membrane, interdigitation can relieve the stress caused by the increased steric hindrance.
Pressure-induced interdigitation is dependent on lipid hydrocarbon chain length and the chemical structure, much like chemically-induced interdigitation. Ether- and ester-linked lipids with longer chains require less pressure to interdigitate [51,54]. Under high temperature and pressure conditions, ester-linked lipids behave similarly to the equivalent ether-linked lipids at normal pressure . Pressure-induced interdigitation is not universal, however. As with chemically-induced interdigitation, certain lipids do not interdigitate even under high pressure [34,55,56].
2.4. Spontaneous interdigitation in ether-linked lipids and 1,3-DPPC
The type of bond that connects the hydrocarbon chain to the lipid head group also affects the thermodynamic properties. Switching either or both of the ester bonds of DPPC with ether linkages results in a small increase in the
The similarity of DHPC to DPPC also allows for the comparison between ether- and ester-linked lipids. It is consistently easier to interdigitate ether-linked lipids whether through chemical means [17,48,49] or by the application of pressure [51,61]. Furthermore, the ether-linked 1,2-di-
While the majority of PC lipid studies use lipids with the hydrocarbon chains on the
2.5. The monofluorinated analogue of DPPC: F-DPPC
The monofluorinated analogue of DPPC, 1-palmitoyl-2-(16-fluoropalmitoyl)
FTIR spectroscopy. It is possible that this relates to a conversion from interdigitated to non-interdigitated gel right before the transition into the liquid crystalline phase . The main transition is also characterized by a large main transition hysteresis (Figures 8 and 9) [67,69]. Additionally, the
It appears that the fluorine must be located on the terminal hydrocarbon chain to have a dramatic effect on interdigitation. When the fluorine substitution is not located on the terminal carbon, DSC data reveal that the physical properties are only modestly changed and they are largely miscible with the non-fluorinated parent lipid . Lipids with more fluorine, such as when the 13-16 carbons are perfluorinated, do not spontaneously interdigitate either [71,72]. Therefore, it is the interaction of the polar terminal C-F bond with the aqueous interface that encourages interdigitation . The large dipole moment is the most likely culprit for stabilizing the interdigitated phase by reducing the unfavorable exposure of the hydrophobic acyl chains to water. However, the slightly larger van der Waals radius and the possibility of weak hydrogen bonding may also play a role [73-77].
2.6. The interdigitated gel phase in anionic lipids
As with PCs, di-saturated long chain phosphatidylglycerols (PGs) have a strong propensity towards interdigitation (Table 3) . The negatively charged PGs are commonly found in microbial membranes . The interaction of some peptides with lipids is heavily dependent on the composition of the membrane . This contributes to the ability of antimicrobial peptides to selectively target microbial membranes . Recently, it was found that DPPG has the ability to form a quasi-interdigitated gel phase with the addition of the human multifunctional peptide LL-37 [81,82]. The antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) has a similar effect below the main transition temperature of saturated PGs . In these instances, the peptide shields the acyl chains of the interdigitated lipid from the aqueous layer by orienting in the interfacial region below the
Furthermore, other chemicals such as Tris-HCl induce interdigitation in DPPG by binding between lipids, resulting in the increased area per head group that favors interdigitation . As in zwitterionic lipids, interdigitation relieves head group repulsion in charged lipids by allowing for a larger area per head group . Charge repulsion in DPPG leads to tilted acyl chains in the non-interdigitated bilayer . This is similar to the ethanol-induced interdigitation of DPPC, where the increased head group size increases the tilt in the gel phase and which ultimately results in the interdigitated gel phase . Ethanol further enhances interdigitation in DPPG, most likely by partitioning into the interfacial region and reducing the exposure of the terminal methyl groups to water .
|myelin basic protein|||
|choline and acetylcholine|||
When ethanol substitutes for water in the transphosphatidylation reaction catalyzed by phospholipase D, phosphatidylethanols (Peth) are formed [84,92]. Peth lipids are unique because they have a small anionic lipid headgroup (Figure 2). These lipids are biologically relevant since Peths accumulate in membranes of animal models of alcoholism . Like DPPG, DPPeth can be chemically induced to interdigitate with Tris-HCl and the interdigitated phase is stabilized with the addition of ethanol .
2.7. The interdigitated gel phase in cationic lipids
Cationic lipids with modified head groups can spontaneously form interdigitated gel phases below the main transition. One recent example is the positively charged lysyl-DPPG, which is DPPG with a lysine moiety attached. Lysyl-DPPC forms an interdigitated phase primarily due to the large repulsion between head groups .
Another modification is the esterification of the phosphate head group, which increases the steric bulk and changes the molecule from zwitterionic to positively charged, allowing interdigitation [95,96]. For example, the P-O-ethyl ester analogue of DPPC, 1,2-dipalmitoyl-
Vesicles formed from cationic triester lipids readily fuse with anionic lipids . This may help explain why lipoplexes made from cationic
In some instances, DNA can be sandwiched between interdigitated gel phase lipid sheets into a rectangular columnar two-dimensional superlattice [97,99]. The gel-to-liquid crystalline phase transition results in the contraction of the DNA strand arrays so that the mean charge density is balanced with the increased positive charge of the non-interdigitated lipid. Additionally, in the non-interdigitated liquid crystalline phase, the interlamellar correlation in DNA ordering is no longer observed .
Phosphatidylethanolamines (PEs) are distinct due to their strong reluctance to interdigitate. PEs are not susceptible to alcohol-induced interdigitation  or pressure-induced interdigitation . Even the ether-linked DHPE does not interdigitate with pressure [34,56]. A major reason for this is that the PE headgroup can form hydrogen bonds . PC headgroups interact through a weaker electrostatic attraction between the positively charged quaternary nitrogen and the negatively charged oxygen of a neighboring lipid headgroup . Additionally, the smaller size of the PE headgroup also allows for closer interaction (less repulsion) [2,8,102].
2.9. Unsaturated phospholipids
Unsaturated lipids with common head groups and acyl chains of nearly equal length are strongly disfavored to interdigitate spontaneously. In general, unsaturated lipids are also resistant to both pressure- and chemically-induced interdigitation [44,103,104]. Even under high pressure, unsaturated lipids typically retain the transition from the non-interdigitated lamellar gel phase (
Furthermore, unsaturated lipids have substantially lower main transition temperatures [7,10]. As interdigitation is highly unfavorable in the liquid crystalline phase, the relevant temperature range of the gel phase where interdigitation is likely to occur is much smaller. The main transition tends to be lowered the most when the double bond is located near the middle of the fatty acid chain [2,10,36]. Although double bonds that are
The inhibition of interdigitation also applies to lipid mixtures involving unsaturated lipids. In a model membrane of DPPC/DOPC/ergosterol, increasing the unsaturated lipid or sterol component co-operatively hinders the formation of the interdigitated phase . DOPC is known to result in a more disordered and less tilted gel phase and can lead to phase separation at higher concentrations . As a consequence, it was hypothesized that changes in the plasma membrane composition may play a role in the ethanol tolerance of yeast cells during fermentation ( and references therein).
There are some exceptions, however. While lipids with double bonds on both chains are particularly unlikely to interdigitate, there are a few examples of interdigitation where only one chain has a double bond. For example, McIntosh et al. tested the ethanol-induced interdigitation of five positional isomers of 1-eicosanoyl-2-eicosenoyl-
Therefore, it can be concluded that while it is possible to induce the interdigitated gel phase in unsaturated lipids, they are more resistant to interdigitation compared to the equivalent saturated lipid. Additionally, when the interdigitated phase does occur in unsaturated lipids, the phase appears to be less stable and less ordered than in saturated lipids .
2.10. Membrane curvature and interdigitation
The curvature of the membrane due to the macromolecular size and shape affects the thermodynamic properties . For instance, on DSC scans, small unilamellar vesicles (SUVs) have lower enthalpic peaks and greater widths compared to multilamellar vesicles (MLVs) [2,3]. SUVs also have more mobility and less order in the hydrocarbon chains .
The degree of membrane curvature also affects the ability to interdigitate. Bending in the membrane causes increased steric interference in opposing lipid monolayers . As a consequence, ethanol-induced interdigitation is dependent on curvature, with the more highly curved vesicles requiring more ethanol to interdigitate [111,112]. Sonicated DPPC SUVs are not stable in the presence of ethanol above the threshold concentration for interdigitation . Furthermore, SUVs have a tendency to fuse into large unilamellar vesicles (LUVs), which have properties more similar to MLVs [36,113]. The more planar MLVs allow interdigitated lipids to slide by each other with low steric interference and therefore have the lowest threshold concentrations [44,112].
2.11. Inhibition of the interdigitated gel phase by cholesterol
Just as there are chemicals that induce interdigitation, there are chemicals that inhibit the formation of the
|Interdigitated Lipid System||References|
|3:7 ratio of 16:0 LPC:DPPC|||
In non-interdigitated membranes, cholesterol increases the fluidity of the gel phase, broadens the main transition, and decreases the main transition enthalpy . Figure 12 demonstrates that these effects are also seen in membranes where cholesterol eliminates the interdigitated phase [115-117]. The amount of cholesterol required to prevent interdigitation is related to the stability of the
There are multiple reasons why cholesterol-rich membranes disfavor interdigitation. For example, lipid head group crowding is mitigated by cholesterol serving as a spacer between lipids . If cholesterol is placed within an interdigitated membrane, the increased spacing also increases the likelihood that the terminal lipid methyl groups will be exposed at the aqueous interface. Since the interdigitated phase lacks the thick membrane midplane region of non-interdigitated membranes, hydrophobic cholesterol located within the interdigitated phase is more likely to come in contact with water . Furthermore, cholesterol significantly disrupts the lattice structure of gel phase lipids [108,117,120]. Lastly, in the interdigitated phase of highly asymmetrical lyso-lipids, cholesterol can take the place of the missing acyl chain thereby compensating for the size mismatch between the head group and the hydrocarbon chains [118,121].
2.12. Chemical inhibition of the interdigitated gel phase
In a general sense, solvent inhibitors of interdigitation work in the opposite fashion as chemical inducers. Some researchers have focused on the difference of how kosmotropic and chaotropic solutes interact with lipid membranes [122-124]. Kosmotropes deplete the solution at the interface and increase the interfacial tension whereas chaotropes accumulate in the interface and decrease surface tension . The changes in the structure of water due to these types of chemicals can be attributed to alterations in the hydrogen bonding network of water [126,127]. Kosmotropic substances are classified as water-structure makers, meaning that they stabilize the structure of bulk water. When kosmotropes interact with hydrated lipids, they tend to reduce the interfacial area and inhibit interdigitation . Chaotropic chemicals are classified as water-structure breakers and increase the surface area per lipid, favoring interdigitation [122,124].
The differences between chemicals that induce or inhibit interdigitation have also been illustrated according to the interaction free energy of the lipid membrane interface with solvents ( and references therein). The solvent free energy relationship can be further split into interactions with the polar head groups and interactions with the hydrophobic lipid chains. In this model, when “good” solvents are added, the interfacial area swells to increase the total contract with the solvent. For organic solvents that are water-miscible and have a high solubility for alkanes, such as acetone and ethanol, the interaction increases the interfacial area by reducing the interaction free energy between the solvent and the interfacial alkyl chains . On the other hand, the interaction of “poor” solvents with lipids is unfavorable and has larger free energy penalty. As a result, the interfacial segments shrink in size to prevent contact with the solvent. Consequently, “good” solvents will favor interdigitation while “poor” solvents will destabilize the
The inhibition of interdigitation has also been described in terms of osmotic stress. Chemicals that apply osmotic stress, such as poly(ethylene glycol) tend to inhibit interdigitation . As in the other models described above, this has been proposed to occur because of a decrease in the repulsive interaction between the lipid head groups.
Dimethyl sulfoxide (DMSO) is an example of a solvent inhibitor of interdigitation. The interaction of DMSO with membranes is of great interest because it can be used as a cryoprotectant for biological material, such as stem cells . DMSO can also enhance the permeability of membranes . The mechanism by which DMSO inhibits interdigitation is by decreasing the repulsion between head groups . The ability of DMSO to form unusually strong hydrogen bonds may explain this effect . This phenomenon can be clearly seen in the phase behavior of DHPC. Just as chemicals that favor interdigitation shift the pre-transition of DHPC to a higher temperature; factors that disfavor interdigitation shift the pre-transition to a lower temperature. The suppression of the pre-transition clearly demonstrates that DMSO destabilizes the
A major caveat with these solvent models is that the interactions with lipids are often concentration-dependent. For instance, in the DPPC/DMSO/water system, three distinct effects are found within different DMSO concentration ranges . Perhaps the most remarkable is at above mol fractions of ~0.9 DMSO, the
The disaccharide trehalose is another example of a chemical inhibitor of interdigitation . Like DMSO, the interactions of trehalose with membranes show promise in the cryopreservation of biological material . In the yeast
2.13. The interdigitated gel phase versus the inverted hexagonal phase
A clear inverse relationship exists between the interdigitated phase gel phase and the inverted hexagonal phase (HII) [56,128]. The major structural factor is the relative size of the lipid headgroup and the attraction/repulsion between headgroups. A lipid that forms the inverted hexagonal phase is unlikely to interdigitate and vice versa. The temperature dependence of these phases is also opposite. For example, with DHPC, the interdigitated phase is present only below the pre-transition. The interdigitated phase requires predominately
This relationship also extends to environmental factors that encourage or discourage interdigitation (Table 5). Chemicals that favor the interdigitated phase such as ethanol tend to destabilize the HII phase [128,124 and references therein]. Interdigitation is favored because the surface area per lipid head group in the
|Large head group repulsion||Small head group repulsion|
|Chaotropic chemicals||Kosmotropic chemicals|
|High hydrostatic pressure||Low hydrostatic pressure|
|Low Temperatures||High Temperatures|
2.14. Influence of hydration and pH on the
While most interdigitated systems are studied in excess water, interdigitation can be affected at less than full hydration. For instance, interdigitation of DHPC is reliant on hydration, as coexisting interdigitated and non-interdigitated phases are found at low hydration [142,143]. However, the cationic EDPPC may be interdigitated in the dry state .
Furthermore, substituting deuterium oxide (D2O) for water slightly disfavors the spontaneous interdigitated phase of DHPC . Using D2O also increases the threshold concentration for the chemically-induced interdigitation of DPPC  and increases threshold pressure for interdigitation . These phenomena are explained by the different hydrophobic interactions and interfacial energies in H2O versus D2O [27,144,145].
Changing the pH of the aqueous solution can also affect interdigitation. In DHPC membranes a low pH will inhibit interdigitation . As the pH is lowered the phosphate groups are protonated and ultimately the total repulsive force between head groups is decreased, disfavoring interdigitation . The pH is also highly relevant to the interdigitated phase in charged lipids, such as PGs. At a high pH, the electrostatic repulsion between head groups that encourages interdigitation in PGs is increased .
2.15. Lipid mixtures and interdigitated/non-interdigitated gel phase coexistence
Under certain circumstances, interdigitated and non-interdigitated phases can coexist within a membrane even though the boundaries between these domains are considered to be energetically unfavorable [115,116]. The uneven structure between these domains can significantly increase the membrane permeability [146,147]. With the variety of lipids now known to interdigitate, there are many possible lipid systems that will have complex phase diagrams involving the
Gel phase coexistence can often be found in binary mixtures of a lipid that can spontaneously interdigitate (e.g. F-DPPC or EDPPC) and one that cannot (e.g. DPPC or PE lipids). For example, at equimolar amounts of F-DPPC and DPPC, interdigitated F-DPPC-rich domains create a phase-segregated system [69,117]. On DSC scans this manifests itself as multiple peaks (Figure 8). The peaks with the greatest transition hysteresis likely correspond to interdigitated domains rich in F-DPPC. When the F-DPPC molar fraction is large, the hysteresis is also increased . Additionally, gel phase coexistence occurs in the mixture of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) and EDPPC . However, this is not true of all such binary mixtures. Outside of the phase transition regions in DPPC/EDPPC, for instance, there is no gel phase segregation .
Mixing an interdigitated lipid with cholesterol can also produce gel phase coexistence. Cholesterol-poor interdigitated domains and cholesterol-rich non-interdigitated domains have been found in DHPC/cholesterol , F-DPPC/cholesterol , and EDPPC/cholesterol . For these mixtures, the lipids with the most stable interdigitated phase tend to have a larger region of phase coexistence within the phase diagram.
Alternatively, a lipid such as DPPC that can be chemically induced to interdigitate can be mixed with lipids that cannot, such as PE lipids . The DPPC-rich domains will interdigitate with ethanol, but domains composed of mostly PE lipid will not. A similar result can be achieved in mixtures of DPPC/cholesterol/ethanol, where the cholesterol-rich domains remain non-interdigitated in the presence of ethanol [39,146].
It is also possible to have coexistence in membranes with only one lipid. For instance, coexisting interdigitated and non-interdigitated phases form in supported F-DPPC membranes where the lateral expansion of the lipid film is restricted . This results in a “frustrated” state, where the energetically favorable interdigitated phase cannot fully form due to constraints in topology and the available surface area . Additionally, while the 16-carbon chain length DPPG does not spontaneously interdigitate, the 18-carbon chain DSPG spontaneously forms an interdigitated gel phase that coexists with a non-interdigitated gel phase . This two-phase coexistence was attributed to a kinetically trapped system that is not at thermal equilibrium .
2.16. Applications of the interdigitated gel phase
One of the most promising applications for the interdigitated gel phase is the creation of large unilamellar vesicles termed interdigitation-fusion (IF) vesicles [44,148]. Figure 14 demonstrates the process for the creation of IF liposomes using ethanol . Below the main transition, the ethanol causes the formation rigid and flat interdigitated sheets . These sheets are surprisingly stable under the
The IF procedure can also be used to create multicompartment vesicle-in-vesicle structures called “vesosomes” . These multicompartment vesicles should be closer replicas of eukaryotic cells than regular vesicles [150,153]. Therefore, vesosomes have the potential to more closely mimic biological conditions and reactions in artificial cells [150,154,155]. Furthermore, the retention of encapsulated material can be substantially increased in vesosomes [151,156,157]. These vesicles are highly customizable because the composition of the inner and outer components can be varied [149,150,154]. As a result, it is theoretically possible to use vesosomes as controlled nanoreactors [153,155]. For complex and expensive chemistry such as enzyme reactions, vesosomes should be able to optimize reaction conditions and drastically reduce the amount of reagents needed .
As described by Ahl et al. , there are four general guidelines for IF liposomes: (1) the lipids must be able to form the interdigitated phase; (2) the precursor liposomes should be small, preferably sonicated SUVs; (3) the temperature of the precursor SUV suspension after the addition of the alcohol must be below the
A similar result can be achieved using pressure to create pressure-induced fusion (PIF) liposomes . An advantage of this technique is that no organic solvent is required and it is an effective sterilization method . The captured volume of the IF or PIF vesicles is larger than other techniques for liposome preparation ( and references therein).
As an analytical instrument, DSC offers many advantages. One advantage is the simplicity of the sample preparation procedure. Samples do not have to be supported or spatially oriented and do not require the insertion of a membrane probe. For sensitive low enthalpy phase transitions, it is a great benefit not to need a probe so that the purity of the sample can be maintained. The importance of this can be seen in alcohol-induced interdigitation, where the low enthalpy pre-transition is an important aspect of the analysis (Figure 5) . Moreover, the effects of pressure can be measured concomitantly with calorimetry data with the appropriate equipment. This greatly expands the range of the phase diagram that can be experimented with.
We have shown that DSC can accurately measure changes in the thermodynamic properties of phospholipid membranes with the addition of chemicals that either encourage or discourage interdigitation. DSC is particularly well-suited for the study of chemically-induced interdigitation because it is sensitive enough to detect small, incremental changes in phase transition temperatures (Figure 5). With the capability to perform heating and cooling scans at a constant rate, the transition hysteresis can also be easily determined. In addition, the transition enthalpy can highlight the “biphasic” behavior above and below the threshold concentration for interdigitation (Figure 6).
Moreover, DSC can reveal how changes in either the hydrocarbon chains (Figure 1) or in the polar head group (Figure 2) will affect the thermodynamics. Modifications that either encourage or discourage interdigitation are summarized in Figure 15. Understanding the importance of structural differences reveals the importance of lipid diversity in biological membranes. Lipid composition can help explain why, for example, a peptide might interact differently with human versus microbial membranes . With the increasing popularity of liposomes for pharmaceutical applications and research, it also is essential to find suitable lipid candidates. For instance, calorimetry can be applied to screen potential IF vesicles by determining whether interdigitation is present and by determining the
In addition, more information can be inferred from DSC data than the phase transition temperature. With careful analysis, the nature of the lipid/solvent interaction and the properties of the chemicals themselves can be derived. For example, the characteristics of kosmotropic and chaotropic chemicals are clearly reflected in their effects on lipid membranes (see section 3.12.). This analysis can also increase the understanding of how chemicals interact with biological membranes, such as why chemicals like DMSO and trehalose can protect cells during cryopreservation .
However, DSC also has limitations when analyzing phospholipid samples. Perhaps the greatest weakness is the lack of direct structural information. As a consequence, relying solely on DSC data can be misleading. For instance, the pre-transition peaks of DPPC and DHPC look similar on DSC thermograms. However, the actual nature of the transition is substantially different (Figure 3). While the structure can often be reasonably inferred from thermodynamic properties, it is not as robust as other experimental techniques . Additionally, while alterations in the macromolecular structure can be reflected in DSC data (see section 3.10.), the changes are not specific enough to be able to infer the true structure.
Overlapping or multiple transitions can also present a problem. In F-DPPC/DPPC, the multiple peaks reflect the presence of phase segregation (Figure 8), but this is not always the case. Multiple DSC peaks can also indicate separate phase transitions that involve the entire membrane. In the case of EDPPC, different morphologies result in separate DSC peaks (Figure 11) . Overlapping peaks can also obscure individual transitions, especially when there are multiple components in the membrane and the transition peaks are broad.
Fortunately, one of the greatest strengths of DSC data is that it is highly compatible with other analytical techniques. In the case of the
The stability of the interdigitated phase plainly demonstrates the balance of forces within the membrane. Factors as varied as electrostatic and steric interactions, van der Waals forces, solvent binding at the interface, and the presence of double bonds all contribute to the properties of hydrated phospholipid membranes. DSC provides a way to judge the resulting balance of these forces by measuring the stability of different thermodynamic phases. Consequently, the wealth of information calorimetric analysis provides ensures that DSC will remain an invaluable tool for the study of membrane biophysics.
differential scanning calorimetry (DSC)
main transition temperature (Tm)
pre-transition temperature (Tp)
small unilamellar vesicle (SUV)
large unilamellar vesicle (LUV)
multilamellar vesicle (MLV)
interdigitation-fusion vesicle (IFV)
interdigitated gel phase (LβI)
planar gel phase (Lβ′)
ripple gel phase (Pβ′)
liquid crystalline phase (Lα)
inverted hexagonal phase (HII)
crystalline bilayer phase (Lc)
1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC or β-DPPC)
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 LPC)
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC or Et-DPPC)