Surfactant-induced membrane dynamics and skin irritation
168
Presented by: Masahiro Takagi
【Background】
There are many surfactant-containing products. Since surfactants may cause skin and eye irritation, they should be evaluated beforehand. Existing evaluation methods include the Draize test, which evaluates the degree of inflammation by dropping surfactants to eyes of rabbits, and the alternative evaluation method by quantifying cell viability after adding surfactants to corneal cells. These methods have the problems of animal welfare and low quantitativity. We have been studying to evaluate the irritation of surfactants by characterizing the membrane dynamics after adding surfactant to biomimetic membranes(1).
【Method】
Cell-sized liposomes composed of unsaturated phospholipids (1,2-dioleoyl -sn-glycero-3-phosphocholine) were prepared, and the membrane dynamics of liposomes after the addition of surfactant was observed using laser confocal microscopy.
Flip-Flop rates of surfactant-containing biomimetic membranes were measured using asymmetric nano-sized liposomes in which only the fluorescent dye of the outer leaflet. The ratio of the fluorescence intensity of the outer leaflet to the total was examined by fluorescence spectrophotometer and the data was fitted to obtain the Flip-Flop rate.
【Result & Discussion】
An increase in excess surface area of liposomes occurred for the addition of strongly irritating surfactants, while they shrank by adding low or no irritating surfactants. Similar membrane dynamics was observed with amino acid surfactants depending on the stimulus intensity. Our method is applicable for mixtures of surfactants.
We observed the membrane invagination and the pore formation for the high-irritating surfactant such as TritonX-100. On the other hand, the large deformation could not be found in the case of non-irritating surfactant such as Tween20 and minimally-irritating surfactants. The deformation was determined by the acquisition of the excess surface area by addition of surfactants. In order to acquire the excess surface area, the surfactant molecules added from outside of liposome should be included in the inner leaflet of bilayers. If the surfactants in the outer leaflet moved to the inner leaflet (flip-flop), the bilayer obtained the excess surface area.
In the case of low- or non-irritating surfactants, the liposomes initially shrank, and the acquisition of large excess area was not observed. Therefore, the flip-flop movement of surfactants hardly occurred. Since the surfactants extracted the lipid molecules as micelles, the total number of molecules in the liposomes was decreased, and the liposomes shrank consequently. The number of surfactants inserted into the outer leaflet, the number of surfactants moved to the inner leaflet (flip-flop), and the number of surfactants excluded from the bilayer as micelles were defined as Vin, Vflip-flop, and Vout, respectively.
We considered that membrane dynamics depends on the rate of Vin, Vflip-flop, and Vout. If we could measure these rates with various surfactants, we could predict the surfactant-induced membrane dynamics and the degree of irritation by the surfactants in future.
In this presentation, we will discuss further the molecular mechanism of membrane dynamics caused by the addition of surfactants based on the Flip-Flop rate.
In addition, the correlation between the results of this evaluation system and the stinging test, a sensitivity test to evaluate itching and tingling sensations in people with sensitive skin, will be presented.
(1) Hamada, T., et al.,
Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome.
J. Phys. Chem. Lett. 2012, 3 (3), 430–435.
There are many surfactant-containing products. Since surfactants may cause skin and eye irritation, they should be evaluated beforehand. Existing evaluation methods include the Draize test, which evaluates the degree of inflammation by dropping surfactants to eyes of rabbits, and the alternative evaluation method by quantifying cell viability after adding surfactants to corneal cells. These methods have the problems of animal welfare and low quantitativity. We have been studying to evaluate the irritation of surfactants by characterizing the membrane dynamics after adding surfactant to biomimetic membranes(1).
【Method】
Cell-sized liposomes composed of unsaturated phospholipids (1,2-dioleoyl -sn-glycero-3-phosphocholine) were prepared, and the membrane dynamics of liposomes after the addition of surfactant was observed using laser confocal microscopy.
Flip-Flop rates of surfactant-containing biomimetic membranes were measured using asymmetric nano-sized liposomes in which only the fluorescent dye of the outer leaflet. The ratio of the fluorescence intensity of the outer leaflet to the total was examined by fluorescence spectrophotometer and the data was fitted to obtain the Flip-Flop rate.
【Result & Discussion】
An increase in excess surface area of liposomes occurred for the addition of strongly irritating surfactants, while they shrank by adding low or no irritating surfactants. Similar membrane dynamics was observed with amino acid surfactants depending on the stimulus intensity. Our method is applicable for mixtures of surfactants.
We observed the membrane invagination and the pore formation for the high-irritating surfactant such as TritonX-100. On the other hand, the large deformation could not be found in the case of non-irritating surfactant such as Tween20 and minimally-irritating surfactants. The deformation was determined by the acquisition of the excess surface area by addition of surfactants. In order to acquire the excess surface area, the surfactant molecules added from outside of liposome should be included in the inner leaflet of bilayers. If the surfactants in the outer leaflet moved to the inner leaflet (flip-flop), the bilayer obtained the excess surface area.
In the case of low- or non-irritating surfactants, the liposomes initially shrank, and the acquisition of large excess area was not observed. Therefore, the flip-flop movement of surfactants hardly occurred. Since the surfactants extracted the lipid molecules as micelles, the total number of molecules in the liposomes was decreased, and the liposomes shrank consequently. The number of surfactants inserted into the outer leaflet, the number of surfactants moved to the inner leaflet (flip-flop), and the number of surfactants excluded from the bilayer as micelles were defined as Vin, Vflip-flop, and Vout, respectively.
We considered that membrane dynamics depends on the rate of Vin, Vflip-flop, and Vout. If we could measure these rates with various surfactants, we could predict the surfactant-induced membrane dynamics and the degree of irritation by the surfactants in future.
In this presentation, we will discuss further the molecular mechanism of membrane dynamics caused by the addition of surfactants based on the Flip-Flop rate.
In addition, the correlation between the results of this evaluation system and the stinging test, a sensitivity test to evaluate itching and tingling sensations in people with sensitive skin, will be presented.
(1) Hamada, T., et al.,
Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome.
J. Phys. Chem. Lett. 2012, 3 (3), 430–435.