Atomic Force Microscopic Analysis of the Effect of Lipid Composition on Liposome Membrane Rigidity

Abstract

Mechanical rigidity of the liposome membrane is often defined by the membrane bending modulus and is one of the determinants of liposome stability, but the quantitative experimental data are still limited to a few kinds of liposomes. Here, we used atomic force microscopy to investigate the membrane bending moduli of liposomes by immobilizing them on bovine serum albumin-coated glass in aqueous medium. The following lipids were used for liposome preparation: egg yolk phosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and N-(carbonylmethoxypoly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. By using liposomes of various compositions, we showed that the thermodynamic phase state of the membrane rather than the electric potential or liposome surface modification with poly(ethylene glycol) is the predominant determinant of the bending modulus, which decreased in the following order: solid ordered > liquid ordered > liquid disordered. By using the generalized polarization value of the Laurdan fluorescent probe, we investigated membrane rigidity in terms of membrane fluidity. Atomic force microscopic analysis was superior to the Laurdan method, especially in evaluating the membrane rigidity of liposomes containing hydrogenated soybean phosphatidylcholine and cholesterol. Positively charged liposomes with a large bending modulus were taken up by cells more efficiently than those with a small bending modulus. These findings offer a quantitative method of analyzing the membrane rigidity of nanosized liposomes with different lipid compositions and will contribute to the control of liposome stability and cellular uptake efficiency of liposomal formulations intended for clinical use.

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid-ordered phase, determined using AFM force spectroscopy

“…Overall, we conclude that milk SM bilayers exhibited dramatically higher Young modulus values (tens of MPa order or magnitude), hence stiffness, when in the gel phase, as compared with the fluid or Lo phases (MPa order of magnitude). The latter phases showed close Young modulus values of a few MPa, but the Lo phase was unexpectedly less stiff than the fluid phase [74].…”

“…Similarly, Liang et al [73] showed that Egg PC bilayers (T > Tm = −15°C) exhibited higher Young modulus when cholesterol was added (i.e., in Lo phase rather than fluid phase). From the analysis of liposomes of several compositions with both fluorescence and AFM force spectroscopy, Takechi-Haraya et al [74] calculated increasing bending rigidities in the order fluid < Lo < gel phase. On the other hand, interfacial elasticity has been shown to be lowest for the Lo phase, when compared to that of liquid-condensed (~gel) or liquid-expansed (~fluid) phases of sphingomyelin monolayers [75].…”

“…In cells, and most importantly in SM-rich cellular "rafts", cholesterol is likely to be implicated in the regulation of membrane curvature, which is important for many cellular events such as membrane fusion, budding, etc. [74,[78][79][80][81].…”

Response to Comment on “A Liposomal System Capable of Generating CO2 Bubbles to Induce Transient Cavitation, Lysosomal Rupturing and Cell Necrosis”

“…Nevertheless, liposomes can reportedly tolerate large osmotic imbalances (e.g., DPPC/Chol liposomes can sustain a pressure difference of 80 atm) . Additionally, the stability of liposomes can be affected by a number of factors, including their physical size, the chemical structures (e.g., polar head groups, fatty acid side chains, degree of unsaturation, and chain length) of the lipids used, and the content of cholesterol incorporated…”

Response to Comment on “A Liposomal System Capable of Generating CO2 Bubbles to Induce Transient Cavitation, Lysosomal Rupturing and Cell Necrosis”

“…[2] Additionally,t he stability of liposomes can be affected by anumber of factors,including their physical size, [2,3] thec hemical structures (e.g., polar head groups,f atty acid side chains,d egree of unsaturation, and chain length) of the lipids used, and the content of cholesterol incorporated. During the purification process,t he as-prepared liposomes are dialyzed against phosphate buffered saline,t herefore at ransmembrane osmotic gradient is generated.…”