Effects of osmotic pressure on the irreversible electroporation in giant lipid vesicles

Abstract

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (kp) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σc) under different Π. The σc dependent kp values were fitted with a theoretical equation, and the corresponding membrane tension (σoseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σoseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σc and Π induced pore formation in the lipid membranes of GUVs.

Quantification of pulsed electric field for the rupture of giant vesicles with various surface charges, cholesterols and osmotic pressures

“…We fixed the osmolarity of glucose solution C out = 375 and 371 mOsmL -1 , and hence the corresponding Δ C 0 = 13 and 17 mOsmL -1 . The detail description of maintaining the osmolarity difference is reported earlier [ 60 ]. The phase contrast images of GUV at E = 300 Vcm -1 under 17 and 13 mOsmL -1 are shown in Fig 7A (i) and 7A (ii), respectively.…”

“…The electroporation in GUVs under various osmotic pressures was reported earlier [ 60 ], where we calculated the membrane tension generated by the osmotic pressures. In present report, we have aimed to quantify the pulsed electric field for the rupture of GUVs with various surface charges, cholesterols and osmotic pressures.…”

“…The osmotic gradient creates lateral tension in the membranes of GUVs. The detail procedure to apply the Π in GUVs has been described in our recent paper [ 60 ]. To avoid the strong adhesion between the GUVs and the surface of glass slide, the chamber was coated with 0.10% (w/v) BSA dissolved in the same buffer.…”

Effect of osmotic pressure on pore formation in lipid bilayers by the antimicrobial peptide magainin 2

“…Later, other researchers also obtained the similar values of membrane tension of GUVs under P using a different method. 29 We can reasonably expect that the AMP-induced pore formation in the GUV membrane and the following evolution of the pore is observed using the osmotic pressure method. This is supported by the fact that large osmotic pressure itself does not induce rupture of GUVs but forms a transient pore with a life time of less than 100 ms. 6 This fact is reasonably explained as follows: immediately after P-induced pore formation the membrane tension greatly decreases because of rapid leakage of the internal solution from the GUV lumen to its outside due to the positive pressure in the GUV lumen according to Laplace's law, and thus, the pore closes rapidly owing to the large line tension of the pore, which is well supported by the theories.…”

A new purification technique to obtain specific size distribution of giant lipid vesicles using dual filtration

“…The unilamellar lipid vesicles such as GUVs were prepared using the well-known natural swelling method that was developed in 1969 [16]. From then many researchers in many laboratories have been using this simple but effective method for preparing unilamellar vesicles [5,24,[28][29][30][31][32][33][34]. For synthesis of lipid membranes of GUVs, 1,2-dioleoyl-sn-glycero-3-phospho-(1 0rac-glycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphochorine (DOPC) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).…”