Enhanced stability of freestanding lipid bilayer and its stability criteria

We present a new strategy to dramatically enhance the stability of freestanding lipid bilayers. We found that an addition of a water in oil emulsion stabilizer, SPAN 80 to a solvent phase guarantees nearly millimeter-scale stable freestanding lipid bilayers. The water permeability, bilayer area, contact angle, and interfacial tension were measured as a function of time and SPAN 80-to-lipid weight ratio (ΦSPAN 80) with several different solvents. Surprisingly, the SPAN 80, instead of remaining in the bilayer, was moved out of the bilayer during the bilayer formation. Also we studied the effect of solvent on freestanding bilayer formation, and found that squalene was the only solvent that was not incorporated into the bilayer. The regime of stable bilayer formation was experimentally determined to be 3/1 < ΦSPAN 80 < 15/1, and we suggest general stability criteria for bilayer formation. This technique and the suggested stability criteria can be potentially helpful to many model membrane-based researches in life sciences, physical sciences and biomedical engineering fields.


Results and Discussion
Interestingly, in all our experiments, the freestanding bilayers remained stable at least for several days without changes in the bilayer area or contact angle for an appropriate range of Φ SPAN80 whereas a droplet immediately coalesced for too low Φ SPAN80 . This implies that the SPAN 80 dramatically enhances the stability of freestanding lipid bilayer 18,[20][21][22][23] . Such enhancement in stability of the freestanding bilayer can be explained by the role of SPAN 80 during the impact of the two monolayers which include the following. First, it modifies the spontaneous curvature; its hydrophobic tail is bulky relative to the hydrophilic head, which induces negative spontaneous curvature. For the bilayer with low stability, a transient pore, which is hydrophilic pore through the bilayer with highly positive curvature, is formed before merging of droplet into sub-phase water. The SPAN 80 with negative curvature plays a critical role for preventing the formation of transient pore to stabilize bilayer 35 . Second, it modifies the interfacial tension. As Φ SPAN80 increases, the interfacial tension decreases, thus it reduces the energetic benefit of droplet coalescence 35 . We also checked other surfactants such as oxidized squalene and docosahexaenoic acid (DHA) that have similar molecular shapes significantly enhance the stability of lipid bilayers. In the previous study, this level of stability was achieved only when the limited kinds of lipid (e. g. 1,2-diphytanoyl-sn-glycero-3phosphocholine, DPhPC) with exceptionally bulky tail or solvent (e. g. hexadecane) remaining in the bilayer after zipping process are used, and this result indicates that the SPAN 80 dramatically enhances stability of the freestanding lipid bilayer 18,[20][21][22][23] .
It was expected that our bilayers would be composed of a mixture of lipid and SPAN 80. However, we surprisingly found that SPAN 80 is likely to be moved out of the bilayer during/after the bilayer formation. To systematically verify the removal of SPAN 80 from the bilayer, we measured the bilayer area, contact angle, bilayer tension, and adhesion energy of two monolayers of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC). We performed all of our experiments at 25 °C where both lipids exhibit a liquid disordered phase 36 . Figure 2(a,b) is the plot of the bilayer area and the contact angle of the freestanding bilayer as a function of time. At t = 0, the bilayer area is of the same diameter d ≈ 220 μ m for both DOPC and DMPC. For DMPC, a drastic change in the bilayer area and the contact angle (θ) occurs at t < 200 sec, followed by the constant values d = 523 μ m and θ = 56°, whereas for DOPC, the bilayer area and contact angle remain unchanged. Figure 2(c) shows the interfacial tension of the bilayer γ B of DOPC and DMPC at Φ SPAN80 = 5/1. The bilayer interfacial tension  The increase in the adhesion energy over time suggests that the bilayer composition changes after bilayer formation. When only SPAN 80 was used without any lipid, no adhesion was observed, implying that zero adhesion exists between SPAN 80 molecules. Therefore, to maximize the adhesion (to lower the energy), lipid molecules should go into the bilayer, excluding SPAN 80 out of the bilayer. At the same time, this demixing process of lipid and SPAN 80 results in an entropic penalty, more specifically, the entropy of mixing. In other words, the competition between adhesion energy and the entropy of mixing determines the distribution of SPAN 80. For the DOPC and DMPC bilayer at Φ SPAN80 = 5/1, the estimated entropic penalty of SPAN 80 is at most in the same order of magnitude as the energetic gain obtained by introducing more lipids in the bilayer region (see Supplementary Figure S3). Therefore, the decrease in the bilayer interfacial tension for DMPC at an early stage (t < 200 sec) in Fig. 2(c) supports that SPAN 80 is removed from the bilayer, to increase adhesion between the two monolayers, as seen in Fig. 2(d). During this period, SPAN 80 is removed from the lipid bilayer and the interfacial tension of the DMPC bilayer drops into the plausible range, when compared with the bilayer rupture tension γ br (DMPC) ≈ 2.7 mN/m (at least, the bilayer tension should be smaller than the rupture tension). γ B of DOPC (4.3-5.7 mN/m) is also less than γ br (DOPC) ≈ 10.2 mN/m 38 .
Another evidence that SPAN 80 is likely to be removed from the bilayer is shown in Fig. 3, the water permeability measurement. The 100 mM NaCl dissolved in the bottom water of the plane interface generates osmotic gradients across the bilayer, resulting in water transport through the lipid bilayer membrane (Fig. 3(a)). We measured the volume change in the water droplet as a function of time 19,20 . In Fig. 3(b), the water permeability of both the DMPC and DOPC bilayer at Φ SPAN80 = 5/1 decreases from 1521.3 μ m/sec (DMPC) and 169.7 μ m/sec (DOPC) to reach constant values of 83.0 ± 6.0 μ m/sec (DMPC) and 103.6 ± 4.2 μ m/sec (DOPC) after the bilayer formation. This equilibrium permeability is in good agreement with the previous measurements: 83 ± 7.6 μ m/sec for DMPC, 56 ± 9 and 158 ± 5.8 μ m/sec for DOPC 39,40 . The initial decrease in water permeability is consistent with the adhesion measurement and thus is most likely due to the process of removing SPAN 80 from the bilayer. Moreover, this initial decrease in water permeability is similarly shown for different stabilizer, squalene oxide, and values of the equilibrium permeability are almost identical (102.6 ± 6.0 μ m/sec for squalene oxide) no matter what kind of stabilizer is used. This suggests that the freestanding bilayer at equilibrium might be composed of DOPC (or DMPC) lipid only.
The permeability result (Fig. 3(b)) also implies that our freestanding bilayer is squalene-free since this is consistent with the measurement for lipid vesicle that has no solvent in it. It is also widely known that squalene does not invade into bilayers or in between two monolayer leaflets [6][7][8][9]21,22 . Decane and hexadecane exhibit a lower permeability in comparison with squalene. A previous study reported that decane and hexadecane remain in the lipid bilayer after the formation of DIB 21,22 . When the bilayer contain a solvent such as decane or hexadecane, water molecules will cross the solvent layer in addition to the lipid bilayer, which results in drops in the water  Figure S5). We note that the interfacial tensions of both decane and hexadecane are around the rupture tension of the DOPC bilayer (≈ 10.2 mN/m) in the absence of solvents.
Combining all the results above, we set up stability criteria for SPAN 80 stabilized bilayer formation (Fig. 4). At very low Φ SPAN80 , SPAN 80 does not reduce the interfacial tension enough to form a stable bilayer, and thus coalescence immediately occurs as soon as a droplet is in contact with the planar surface. For sufficiently high Φ SPAN80 , the interfacial tension is low enough for stable bilayers, exhibiting the successful zipping process with the intermediate contact angle between 90° and 180°. For very high Φ SPAN80 (in a case of 2γ M < γ B ), however, the contact angle reaches 180°, and the adhesion does not occur. Even if the contact angle does not reach 180°, too high Φ SPAN80 reduces adhesion between the two monolayers, and in this case, the entropy of mixing is too big to increase the adhesion, leaving some SPAN 80 in the bilayer. Moreover, the regulation of interfacial tension directly affects the three phase contact angle of the lipid bilayer: two lipid monolayers and a bilayer. The importance of the interfacial tension regulation is easily seen in the DMPC bilayer formation. For DMPC, the interfacial tension of the lipid bilayer is nearly zero. The DMPC monolayer interfacial tension is also low enough, so it appears to form a stable bilayer at first. However, the contact angle of the lipid bilayer changes over time, and eventually becomes very low (< 60°), and the abrupt change at the kink seems to make the bilayer unstable. The stability of the DMPC bilayer becomes worse if the contact angle is very low. Therefore, to enhance the stability of the bilayer and to simultaneously obtain solvent-free and SPAN 80-free bilayers, there is an appropriate and optimum range of Φ SPAN80 . Since different lipid species show different lipid bilayer interfacial tension, to form stable freestanding lipid bilayer this proper range of Φ SPAN80 will change. We also should note that previous DIBs use higher concentrations of lipids that might have an appropriate interfacial tension [18][19][20][21][22][23] .

Conclusion
We demonstrated a new strategy to dramatically enhance stability of DIB with a large area, planar and solvent-free as well by using W/O emulsion stabilizer, SPAN 80. Surprisingly, SPAN 80 is most likely to be moved out of the bilayer, maximizing the adhesion of the lipid monolayers, and overcoming the entropy of mixing penalty. This removal of SPAN 80 was demonstrated by time-dependent adhesion and permeability experiments. We also showed that the freestanding bilayer fabricated by our technique is squalene-free as well. We finally suggested stability criteria for the SPAN 80 stabilized freestanding bilayer formation, involving the regulation of interfacial tension by controlling SPAN 80 concentration. This stabilization strategy can be universally applied to various freestanding bilayer formation techniques such as the conventional DIBs and the traditional black lipid membranes.

Methods
Dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC) and SPAN 80 are purchased. Squalene oxide is prepared by direct light exposure on squalene for four days with air contact. We use deionized water for all of our experiments. The imaging experiments were performed by using homebuilt side-view microscope. The sample of phospholipid (DMPC or DOPC) in chloroform is contained in glass vial and dried in vacuum. SPAN 80 dissolved in squalene is added into the dried phospholipid, and then sonicated for 30 minutes. We prepare a trough filled with water, and the phospholipid solution is placed on top of water to form a planar squalene/ water interface. The glass capillary of 0.78/1.0 mm in inner/outer diameter respectively is tapered to 10 μ m of diameter by a micropipette puller. The capillary is filled with water and then mounted to the micro-injector. The capillary tip is placed above the squalene/water interface. By applying a pressure of ~100 hPa, the droplet of ~300 μ m diameter is introduced right above the planar interface. Both planar and droplet squalene/water interfaces are incubated for over 10 minutes for the adsorption of phospholipid and SPAN 80 monolayers, which are termed as planar monolayer and droplet monolayer, respectively. The droplet is moved toward the planar interface until the droplet gently touches the planar interface. After a few minutes of waiting, two monolayers undergo "zipping" process, in result, form the lipid bilayer between two water phases. The size of freestanding lipid bilayer can be controlled by adjusting the droplet size. Further details of monolayer interfacial tension measurement, water permeability measurement, and adhesion energy measurement are summarized in the Supplementary Information.