Membrane permeabilization and shape transition: role of asymmetry and depth of modification PMMH

Stéphanie Bonneau1, Julien Heuvingh2, Halina Mojzisova1, Daniel Brault1

Paris 7 – CNRS UMR7636

The oxidation of unsaturated lipids is of great interest, both from the biological and medical points of view. The photochemical induction of oxidation processes is an effective and clean way of inducing oxidation, allowing fine control of both initiation and orientation. It is supported by the ability of certain molecules, called photosensitizers (PS), to generate ROS upon light irradiation. In this work, we first attempt to correlate the depth of the peroxidation within the membrane and the resulting photo-induced membrane permeabilization. We focus on Chlorin e6 a tricarboxylic photosensitizer in comparison with the neutral hydrophobic m-THPC (Foscan®). Different approaches to evaluate the photo-induced lipid peroxidation and the membrane permeation the photoinduced are presented. In a second time, we photochemically induce the lipid oxidation in DOPC-GUV. th photosensitizer used, Ce6, allows a symmetric as well as an asymmetric targeting of the membrane bilayers and a fine control of the location of the oxidation. The induced morphological transitions are corelated to the targeted leaflet and shows a decrease in the spontaneous curvature of the targeted leaflet. The eventual permeabilization of the membrane have been measured and can be linked to the tension due to the budding of vesicles.

OOH

Peroxide

Lipid

Cleaved-lipid

Photo-peroxidation of DOPC

Depth of the photochemical modification as a determinant of permeabilization efficiency Ce6 and m-THPC singlet oxygen and quantum yields measured by reaction with DPBF are very close. We have measured some photodynamic parameters both in homogeneous solution and in model membranes (DOPC SUV - Small Unilamellar Vesicle).

Chlorin e6 and m-THPC differ by the nature of their lateral chains. Taking into account their structures the Ce6 is most likely located near the interface of the membrane with water, while the m-THPC is burried deeper into the membrane hydrophobic core.

Ce6

m-THPC Lipid peroxidation-diene formation

Membrane permeabilization

The photo-peroxydation of methyl linolate (ML) induces the formation of conjugated dienes and can be monitored by following the absorption at 234nm.

The photodestabilization of the bilayer structure was studied by using carboxyfluorescein (CF) as a model of entrapped molecule.

Within the membrane

In solution

At high concentration the fluorescence of this hydrophilic probe is self-quenched.

Encapsulated fluorophore PS within the membrane

Proportional to 

m-THPC >Ce6

Ce6>m-THPC

The peroxidation is clearly influenced by the relative position of the PS and the substrate of singlet oxygen. Accordingly, for the oxidation of hydrophobic methyl linoleate the m-THPC is more efficient. For Ce6 that is located near the membrane surface, both the poorer colocalization between the substrate and the PS, and the fact that singlet oxygen is more likely to escape into the aqueous phase, reduce the efficiency. However, Ce6 is more efficient to permeabilize the membrane than m-THPC. Our results are not sufficient to understand the photopermeabilization efficiency, and thus we did invastigate the asymmetry effect.

Photoinduced asymmetry of the membrane governs the shape transitions and the permeabilisation Giant Vesicles were asymmetrically labeled with chlorin. The chlorin e6 molecules were either present outside the vesicle, inside the vesicle or both outside and inside. For chlorin present outside the vesicle, chlorin was diluted in the glucose solution before mixing with GUV. For chlorin present inside the vesicles, GUV were prepared with a sucrose solution containing chlorin in the chamber and subsequently rinsed. GUV with symmetrical chlorin distribution were prepared with a sucrose solution containing chlorin in the chamber and then mixed with a chlorin-containing glucose solution.

Chlorin e6 in the outer leaflet of the membrane

Prolate vesicle

10 µm

Pear shape

2 seconds / picture

10 µm

time (s)

Budding

Shape transition and changes in effective spontaneous curvature out

2

11

12

13

Invagination

14

15

28

permeabilization

45

Short time deformation

70

c=c0 exp(-(t-t0)/τ)

Normalized contrast

Chlorin e6 in the inner leaflet of the membrane

out

Ce6 outside

Ce6 both (in/out)

time (s)

Ce6 inside

C0 in

in

Oxidation of the inner leaflet

Oxidation of the external leaflet

Orientation of the shape modifications Negative curvature of the targeted leaflet ADE Model Seifert 1997 Döbereiner 2000

The asymmetrical modifications of the membrane induces a negative curvature of the targeted leaflet. These changes in the spontaneous curvature of the membrane induces subsequently clearly orientated shape transitions From: Seifert 1997; Döbereiner 2000

[Ce6]

[Ce6]

Ce6 outside > Ce6 inside Importance of the asymmetry in the permeabilization process

Conclusions Modification of the spontaneous curvature

Area Volume

Tension of the membrane

Lysis of the GUV

The asymmetrical shape transitions observed in photosensitized GUV reveal changes in their membrane spontaneous curvature. These modifictions are in accordance with the presence of cleaved-lipids by-products of oxidation. Permeabilization and a decrease of lysis tension were also characterized. We developped a model linking the budding due to the spontaneous curvature change to a tension of the membrane up to the lysis level where membranes are permeated. These findings might shed a new light on some membrane permeation phenomenon involved in biomedecine photodynamic approaches and in cell oxidative stress.

Hypothesis : the budding induces a tension of 0.15mN/m