Membrane interactions of lipopeptides and saponins, two

groups of natural

amphiphilesJ. Lorent

(public defence)

Main supervisor

: M-P. Mingeot-Leclercq (FACM)Co-supervisor

: J. Quetin-Leclercq (GNOS)

Introduction : physico-chemical

properties

of amphiphiles

Polar part(hydrophilic)

Apolar

part(hydrophobic)

S

O

O

ONa

Sodium dodecylsulfate

(detergent)

Introduction : selected

amphiphiles of natural

origin

surfactin hederagenin

Saponin Triterpenic

acidLipopeptide

α-hederin

C3C3

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

O

CH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

H

CH2OH

COOH

HO

Isolated

from

Hedera helix

L. Isolated

from

Bacillus

subtilis

HHO H

HO H

HO H

HO H

HO

water (polar)

air (apolar)Interfacial

/ Surface tension or energyγ

= (F / l) or (E / A)

Introduction : amphiphiles (effect

on interfaces)

HHO H

HO H

HO H

HO H

HO

water

air

Interfacial

/surface tensionγ

= (F / l) or (E / A)

Introduction : amphiphiles (effect

on interfaces)

HHO H

HO H

HO H

HO H

HO

water

air

Interfacial

/surface tensionγ

= (F / l) or (E / A)

Introduction : amphiphiles (effect

on interfaces)

HHO H

HO H

HO H

HO H

HO

water

Interfacial

energy

air

Emulsions / suspensions

Introduction : amphiphiles (effect

on interfaces)

• liquid

/ air (foam)

• liquid

(apolar) / liquid

(polar)

creams

Introduction : amphiphiles (self-aggregation)Polar part

(hydrophilic)Apolar

part(hydrophobic)

S

O

O

ONa

water

Polar part(hydrophilic)

Apolar

part(hydrophobic)

S

O

O

ONa

[amphiphile]

Introduction : amphiphiles (self-aggregation)

water

S

O

O

ONa

Polar part(hydrophilic)

Apolar

part(hydrophobic)

[amphiphile] > Critical

micellar

concentration (CMC)

Micelles or other

aggregates

Introduction : amphiphiles (effect

on interfaces)

water

O

H

OH

H

H

O

H

P OO

O

R

O

HS OO

ONa

Introduction : amphiphiles (self-aggregation, intrinsic

curvature

and polymorphism)

Normal hexagonal HI MicellesCone

shape Cubic

(CI

)

ς

> 0Intrinsic

molecular

curvature

Detergents Lysophospholipids

OH

H

HH

Introduction : amphiphiles (self-aggregation, intrinsic

curvature

and polymorphism)

Inverse hexagonal HII Inverted

micellesInverted

cone

shape Inverted

cubic

(CII

)

ς

< 0

Cholesterol

O

H

O

HH

O

H

P OOO

OO

H

N

OH

NH

H

H

O

H

P OO

OR

O

H

Introduction : amphiphiles (self-aggregation, intrinsic

curvature

and polymorphism)

Lamellar

Lα

, Lβ

, Lo (bilayers

or lipid

membranes) Cylinder

shape

ς

= 0

Phosphatidylcholines Sphingomyelins

Introduction : bilayer

dynamics

and cholesterol effects

Lateral

diffusion (10-7s)

Protrusion

(10-9s)

Flip-flop (10-3-104s)

Rotational

diffusion (10-8s)

Trans-gauche isomerization(10-12s)

Bond oscillations (10-12s)

Ondulations (10-6-1s)

Yeagle

et al. (2005)

Gel state Liquid

crystalline

state

Liquid

ordered

state

T

> Tm

Lβ Lα

Lo

+ cholesterol + cholesterol

Introduction : the biological

membrane (lateral heterogeneity

and lipid

rafts)

• enriched

in cholesterol, glyco-sphingolipids, saturated

phospholipids• Variable size (20-200nm)• functional

platforms: receptor

Fas, DISC

assembly

(death

receptor)

Lingwood

(2011)

Lo-phase (liquid

ordered) Lα-phase(liquid

crystalline)

Rafts (Lo

, liquid

ordered

phase)

Introduction : different

cell

deaths

induced

by saponins

cholesterol

mitochondriacaspase-3

condensation, fragmentation nuclei

death

receptor

MPT-pore

cytoplasm

necrosis

caspase-8

saponin

phospholipid

Ca2+

membraneinsertion

Apoptosis

membrane lysis

Ca2+

ROS

cyt

c

Mazzucchelli

et al., 2008

Trump

and Berezesky, 1995

Trump

and Berezesky, 1995

Choi et al., 2008

Jiang et al., 2010

Aim

of the study

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

O

CH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

H CH2OH

COOH

HO

? Raft located

death

receptor

Materials

and methods

: Membrane models•

LUV (large unilamellar

vesicles)/ MLV (multilamellar

vesicles)–

Size = 100 nm-1 µm –

Easy

determination

of lipid

concentration Quantification

of effects

and amphiphilie/lipid

ratio

•

GUV (giant

unilamellar

vesicles)–

Size ~ 10-50 µm–

Difficult

determination

of lipid

concentration Easy

observation

of effects

•

SPB (supported

planar

bilayers)–

Difficult

determination

of lipid

concentration Nanoscopic

effects

•

Cancer cells

(monocytes from

leukemia, U937)–

Cytotoxicity, determination

of cell

death

Light microscopy

GUV

Cryo-TEM

LUV/MLV

AFM

SPB

Cells

Materials

and methods: phase separation

modelsDOPC : DPPC (1:1)

Lα

(liquid

crystalline) / Lβ

(gel state)

Tm

= -3°C Tm

= 42°CTR-DPPE (Lα

)labeled

GUVsDOPC,DMPC

DPPC

Chol

DMPC:Chol

(3:1)Tm

=24°C

Lo Lα Lo

liquid

ordered

/ liquid

crystalline

Interaction of α-hederin

and hederagenin

with

lipids

and

effects

on membrane models

CH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

H

CH2OH

COOH

HO

α-hederin hederagenin

CH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

H

Induction of Highly

Curved

Structures in Relation to Membrane Permeabilization

and Budding

by the Triterpenoid

Saponins, α-

and δ-

hederin

Joseph Lorent1,2, Cécile S. Le Duff3, Joelle

Quetin-Leclercq2

and Marie-Paule Mingeot-Leclercq1*

1Université

catholique de Louvain, Louvain Drug Research

Institute, Cellular and Molecular

Pharmacology, Avenue E. Mounier 73, UCL B1.73.05, B-1200 Bruxelles, Belgium.

2Université

catholique de Louvain, Louvain Drug Research

Institute, Pharmacognosy, Avenue E. Mounier 73, UCL B1.72.03, B-1200 Bruxelles, Belgium.

3Université

catholique de Louvain, -

Institute of Condensed

Matter

and Nanosciences, Molecules, Solids

and Reactivity, Place Louis Pasteur 1, UCL L4.01.04, B-1348 Louvain-la-Neuve, Belgium.

Journal of Biological

Chemistry

2013, 288, pp. 14000-14017

Phase separation

and permeabilization

induced

by the saponin

α-hederin

and its

aglycone hederagenin

in a raft mimicking

bilayer

Langmuir, submitted

Joseph Lorent1,4, Laurence Lins2, Domenech

Òscar3, Joelle Quetin-Leclercq4, Robert Brasseur2

and Marie-Paule

Mingeot-Leclercq1

1Université

catholique

de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, Avenue E. Mounier

73, B-1200 Brussels, Belgium.

2ULg, Centre de Biophysique Moléculaire Numérique, Agro-BioTech

Gembloux, Passage des Déportés, 2, B-

5030 Gembloux, Belgium.

3University of Barcelona, Departament

de Fisicoquímica, Facultat

de Farmacia, UB and Institut de Nanocie ncia

i Nanotecnologia

IN, 08028 Barcelona, Spain.

4Université

catholique de Louvain, Louvain Drug Research

Institute, Pharmacognosy, B1.72.03, Avenue E. Mounier 72, B-1200 Brussels, Belgium.

Interaction with

dehydroergosterol

(DHE) in aqueous

solution : DHE spectroscopy

Both

molecules

bind

to DHE forming

some

type of mixed aggregates

(micelles) I50

(inflexion point) of α-hederin

corresponds to its

CMC (13 µM)

I1

= monomeric

peak(=monomeric

DHE)I2

,I3

= structured

emission(=crystalline

DHE)

340 365 390 415 4400

100

200

300

400

500

600

700increasing [-hederin]

Emission wavelength (nm)

Fluo

resc

ence

inte

nsity

(nm

)

1 10 1000.4

0.6

0.8

1.0

1.2

Log [-hederin] (µM)

Inte

nsity

rat

io (I

1/I 2

)

1 10 1000.4

0.6

0.8

1.0

1.2

Log [hederagenin] (µM)

340 365 390 415 4400

100

200

300

400

500

600

700increasing [hederagenin]

Emission wavelength (nm)

α-hederin hederagenin

I50

= 12.3 ±

3.2

µM I50

= 14.3 ±

8.5 µM

402 396 nm 402 391 nm

I1I2

I2I1

I3I3

TRIS.HCl

(10 mM, pH=7,4)

Permeabilization

of LUV :

cholesterol

dependencePC/SM/PI/Chol

(4:4:3:x)

15 % cholesterol

30 % cholesterol

18h30min

p<0.05, ** p<0.01, *** p<0.001 two-way

ANOVA (18h vs 30min)

-hederin -hederin hederagenin0

25

50

75

100 4

Max

% c

alce

in r

elea

sed

0 10 20 30 40 50

0

25

50

75

100***

***

***

***

**

[-hederin]:[cholesterol]

% c

alce

in r

elea

sed

Permeabilization

depends

on the cholesterol

content and the saponin

sugar

chain

DMPC/Chol

(3:1) [compound] / [lipid] ratio

2 sugars 1 sugar no sugarNumber

of sugars

at

C3:

Calcein

(self-quenching

concentration)

Calcein

assay

LUV

fluorescence

permeabilization

Permeabilization

of LUV :

cholesterol

dependencePC/SM/PI/Chol

(4:4:3:x)

15 % cholesterol

30 % cholesterol

18h30min

p<0.05, ** p<0.01, *** p<0.001 two-way

ANOVA (18h vs 30min)

-hederin -hederin hederagenin0

25

50

75

100 4

Max

% c

alce

in r

elea

sed

0 10 20 30 40 50

0

25

50

75

100***

***

***

***

**

[-hederin]:[cholesterol]

% c

alce

in r

elea

sed

Permeabilization

depends

on the cholesterol

content and the saponin

sugar

chain

DMPC/Chol

(3:1) [compound] / [lipid] ratio

2 sugars 1 sugar no sugarNumber

of sugars

at

C3:

Reorganization

of MLV : cholesterol

dependence

Control

Disruption of bilayer

structure depends

on sugar

chain

and cholesterol

(not shown) Increase

of isotropic

motion and appearance

of hexagonal pattern with

α-hederin

10% α-hederin

20% α-hederin

DMPC/Chol

(3:1)

and macroscopic

phase separation

Malcolm et al. 2003Chemical

shift tensor

of the phosphore atom

31P-NMRPic plus large vesicule

plus grand

micelles

MLV (low

curvature)

(high

curvature)

C1

= highC2

= 0

Reorganization

of MLV : cholesterol

dependence

Disruption of bilayer

structure depends

on sugar

chain

and cholesterol

(not shown) Increase

of isotropic

motion and appearance

of hexagonal pattern with

α-hederin

10% α-hederin

20% α-hederin

Control

DMPC/Chol

(3:1)

+

Reorganization

of MLV : cholesterol

dependence

Disruption of bilayer

structure depends

on sugar

chain

and cholesterol

(not shown) Increase

of isotropic

motion and appearance

of hexagonal pattern with

α-hederin

10% hederagenin

20% hederagenin

Control

DMPC/Chol

(3:1)

>< 100 nm

Sterol/phospholipid

phase separation

in MLV

Decrease

of FRET efficiency

is

significant

only

with

α-hederin

at

high

compound/lipid

ratios

D

0 0.2 0.6 0.8 1.2

F

1

2

3

**

C

I 430/I

371

0 0.25 0.6 0.8 1.21

2

3

*

E

I 430/I

371

0 2500 5000 7500 100001

2

3A

time (s)

I 430/I

371

0 2500 5000 7500 10000

B Control0.250.60.81.2

time (s)

-hederin hederagenin

3h

24h

-hederin/lipids hederagenin/lipids

*p < 0.05, one-way

ANOVA (vs. Control)

[compound]/[lipids]DMPC/Chol

(3:1)

Low

energy

transfert

High energy

transfert

OH

OH

Förster

energy

resonance

transfert (FRET)

DHEDPH-PC

High FRET ratio

Low

FRET ratio

Sterol/phospholipid

phase separation

in MLV

Decrease

of FRET efficiency

is

significant

only

with

α-hederin

at

high

compound/lipid

ratios

D

0 0.2 0.6 0.8 1.2

F

1

2

3

**

C

I 430/I

371

0 0.25 0.6 0.8 1.21

2

3

*

E

I 430/I

371

0 2500 5000 7500 100001

2

3A

time (s)

I 430/I

371

0 2500 5000 7500 10000

B Control0.250.60.81.2

time (s)

-hederin hederagenin

3h

24h

-hederin/lipids hederagenin/lipids

*p < 0.05, one-way

ANOVA (vs. Control)

[compound]/[lipids]

DMPC/Chol

(3:1)

α-hederin hederagenin

10 µM

budding

wrinkled

phase separation

spherical

Incubationtime

Effects

on GUV

< CMC(α-hederin)

2h

48hComplete

transformation of GUV

DMPC/Chol

(3:1)

1hTR-DPPE NBD-DPPE

Fluorescence microscopy

α-hederin hederagenin

10 µM

budding

wrinkled

phase separation

spherical

Incubationtime

Effects

on GUV

< CMC(α-hederin)

1h

2h

48hComplete

transformation of GUV

DMPC/Chol

(3:1)

Phase separation

in GUV

NBD-DPPE (green)TR-DPPE (red)

Confocal

(1 slide)

Confocal

(profile, circumference)

Confocal

(3D)

α-hederin

(10 µM)

Accumulation of mainly

TR-DPPE into

worm-like

domains At

later

stages : separation

of TR-DPPE and NBD-DPPE

TR-DPPE(Lα

)

NBD-DPPE(Lo

)Confocal

slide

Profile (cirumference)

3D reconstruction (all slides)

Confocal

microscopyGUV (DOPC:DPPC:Chol)

Phase separation

in GUV

NBD-DPPE (green)TR-DPPE (red)

Confocal

(1 slide)

Confocal

(profile, circumference)

Confocal

(3D)

α-hederin

(10 µM)

Accumulation of mainly

TR-DPPE into

worm-like

domains At

later

stages : separation

of TR-DPPE and NBD-DPPE

α-hederin hederagenin

40 µM30 min

1h

budding

wrinkled

pore

Incubationtime

> CMC(α-hederin)

spherical

Effects

on GUV

Effects

on GUVα-hederin hederagenin

40 µM30 min

1h

4h

48h

N.D.

N.D.

phase separation

spherical

Incubationtime

Phase separation

in GUV

NBD-DPPE (green)TR-DPPE (red)

Confocal

(1 slide)

Confocal

(profile, circumference)

Confocal

(3D)

Formation of small

circular

domains

with

hederagenin

hederagenin

(40 µM)

Permeation

of GUVs

to FITC-dextran

α-hederin

4 kDa10 µM

Budding(20s)

Gradual

permeation

to FITC-dextran

of 4 kDa

Phase separationControl (90 min)< CMC

(α-hederin)

Dextran-FITC solution

Permeabilization

Permeation

of GUVs

to FITC-dextran

α-hederin

4 kDa10 µM

Budding(20s)

Gradual

permeation

to FITC-dextran

of 4 kDa

Phase separation

(30, 60, 90 min)Control (90 min)< CMC

(α-hederin)

Permeation

of GUVs

to FITC-dextranα-hederin

Immediate

permeation

to FITC-dextran

of 4 kDa

4 kDa

4 kDa

40 µM

10 µM

Budding(20s)

Gradual

permeation

to FITC-dextran

of 4 kDa

Phase separation

No wrinkling(20s) Increasing

wrinkling

Control (90 min)

Control (90 min)> CMC

(α-hederin)

< CMC(α-hederin)

hederagenin

Permeation

of GUVs

to FITC-dextran

Control (48h)

4 kDa40 µM

Permeation

to FITC-dextran

of 4 kDa

after

48h

Phase separation

(48h)

Nanoscopic

effects

of α-hederin

on supported

planar

bilayer

Accumulation of membrane material

into

worm-like

structures (new mesophase) Formation of holes

with

increasing

size upon

time

t = 0 t = 6 min t = 15 min t = 24 min

40 µM

DMPC/Chol

(3:1)α-hederinAtomic

force microcopy

Profile (supported

planar

bilayer)

Nanoscopic

effects

of α-hederin

on supported

planar

bilayer

Accumulation of membrane material

into

worm-like

structures (new mesophase) Formation of holes

with

increasing

size upon

time

t = 0 t = 6 min t = 15 min t = 24 min

40 µM

DMPC/Chol

(3:1)

α-hederin

Conclusions : Membrane interactions of α-hederin

Internal

leaflet

External

leaflet

10 µM < CMC

Budding

Phase separation Transformation intoanother

mesophase

Positive curvature

Hydrophilic

sugar-sugar

interactions

Monomers

Pore formation ?

Transient

defects

?

DMPC

Cholesterol

immediate

later

Internal

leaflet

External

leaflet

40 µM

> CMC

toroidal

macroscopic

pore

immediate

permeabilization

without

transformationinto

new mesophase

(at

least at

short incubation periods)

Conclusions: Membrane interactions of α-hederin

Aggregates

and monomers

DMPC

Cholesterol

Conclusions : Membrane interactions of hederagenin40 µM

> CMC ??

accumulation into

round spots transformation into

inverse structures ? transient

defects

permeabilization

?

Inverse aggregates

?

Investigations on cellsα-hederin

and hederagenin

induce apoptosis and

non-apoptotic cell death in U937 and THP-1 cells in a cholesterol-dependent manner

Joseph Lorent1,2, Joelle Quetin-Leclercq2

and Marie-Paule

Mingeot-Leclercq1*

1Université

catholique

de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier

73, B-1200 Bruxelles, Belgium.2Université

catholique de Louvain, Louvain Drug Research

Institute, Pharmacognosy, UCL B1.72.03, avenue E. Mounier 73, B-1200 Bruxelles, Belgium.

0 20 40 600

25

50

75

100C

time (h)

% c

ell d

eath

0

10

20

30

40A

% fr

agm

ente

d nu

clei

0 20 40 60

D

time (h)

B CTL10 µM15 µM20 µM40 µM

-hederin hederagenin

Cell

death

: Acridine orange/Ethidium

bromide

in U937 cells

Apoptosis

induction = faster

with

α-hederin

than

hederagenin

+ Q-VD-O-Ph

(general

caspase

inhibitor) inhibition of apoptosis

for both

compounds no inhibition of non-apoptotic

cell

death

Non-apoptotic

cell

death

(very

important at

high

α-hederin

concentrations)

Apoptotic(fragmented

nuclei)

Membrane

damage(Ethidium

bromide)

No membrane Damage

(acridine orange)

Non-apoptoticAO/EB staining

0 20 40 600

25

50

75

100C

time (h)

% c

ell d

eath

0

10

20

30

40A

% fr

agm

ente

d nu

clei

0 20 40 60

D

time (h)

B CTL10 µM15 µM20 µM40 µM

-hederin hederagenin

Cell

death

: Acridine orange/Ethidium

bromide

in U937 cells

Apoptosis

induction = faster

with

α-hederin

than

hederagenin

+ Q-VD-O-Ph

(general

caspase

inhibitor) inhibition of apoptosis

for both

compounds no inhibition of non-apoptotic

cell

death

Non-apoptotic

cell

death

(very

important at

high

α-hederin

concentrations)

0 5 7.5 10 12.5 15 17.5 200

10

20

30

***

***

% f

ragm

ente

d nu

clei

0.0 5.0 7.5 10.0 12.5 15.0 17.5 20.0

* *** ******

******

**

Non-depletedCholesterol-depleted24h 48h

Cholesterol

dependence

of total cell

death

/ apoptosis

in U937

Cholesterol

dependence

of cell

death

: decrease

of α-hederin

induced

total celldeath

and apoptosis

in depleted

cells

α-hederin

Non-depleted cells

0 5 10 15 20 25 300

25

50

75

100

******

******

*** *** ***

time (h)

% c

ell d

eath

Cholesterol depleted cells

0 5 10 15 20 25 30

control10 µM15 µM20 µM25 µM

time (h)

two-way

ANOVA*p<0.05, **p<0.01, ***p<0.001vs. depleted

cells

Trypan

blue

staining

(cell

death

assay)

Microscopedeath

cell

Fluorescent DNA intercalator

DAPI (apoptosis

assay)

apoptoticnormal

Cell

nuclei

(fluorescence microscopy)

accumulates

only

in death

cells

0 5 7.5 10 12.5 15 17.5 200

10

20

30

***

***

% f

ragm

ente

d nu

clei

0.0 5.0 7.5 10.0 12.5 15.0 17.5 20.0

* *** ******

******

**

Non-depletedCholesterol-depleted24h 48h

Cholesterol

dependence

of total cell

death

/ apoptosis

in U937

Cholesterol

dependence

of cell

death

: decrease

of α-hederin

induced

total celldeath

and apoptosis

in depleted

cells

α-hederin

Non-depleted cells

0 5 10 15 20 25 300

25

50

75

100

******

******

*** *** ***

time (h)

% c

ell d

eath

Cholesterol depleted cells

0 5 10 15 20 25 30

control10 µM15 µM20 µM25 µM

time (h)

two-way

ANOVA*p<0.05, **p<0.01, ***p<0.001 vs. depleted

cells

Conclusions : Potential

interactions

α-hédcholestérol

mitochondria

cyt

c

Caspase-3

apoptosisDR

MPT

cellnecrosis

Ca2+(*)

*Choi, J.H. et al. 2008**Swamy

and Huat

2003

Caspase-8(*)

membrane lysis

(*)

(*,**)

(*)

Effect of surfactin

on membrane models displaying lipid phase separation

Magali Deleu1°*, Joseph Lorent2°, Laurence Lins3, Robert Brasseur3, Nathalie Braun4, Karim El Kirat4, Tommy Nylander5, Yves F. Dufrêne4

and Marie-

Paule Mingeot-Leclercq2

1Université

de Liège Gembloux Agro-Bio Tech, Unité

de Chimie Biologique Industrielle, Passage des Déportés, 2, B-5030 Gembloux, Belgium

2Université

catholique de Louvain, Louvain Drug Research

Institute, Cellular and Molecular

Pharmacology, Avenue E. Mounier 73, B1.73.05, B-1200, Brussels, Belgium

3Université

de Liège Gembloux Agro-Bio Tech, Centre de Biophysique Moléculaire Numérique, Passage des Déportés, 2, B-5030 Gembloux, Belgium

4Université

catholique de Louvain, Institute of Condensed

Matter

and Nanosciences, Bio and Soft Matter, Croix du Sud 1, L7.04.01, B-1348 Louvain-la-

Neuve, Belgium

5Lund University, Center

for Chemistry and Chemical Engineering, Physical Chemistry, 1S-221 00 Lund, Sweden°Equal First author

BBA, Biomembranes, 2013, 1828, pp. 801-815

Molecular

structure, CMC and activity

of surfactin

CMC = 12.85 ±

0.05 µM

(pH = 8.5)

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

O

(determined

by isothermal

titration

calorimetry)

C13

, C14

, C15

linear

acyl

chain

Tested

on DOPC/DPPC

(1:1) bilayer

at

pH =8,5

0 min 22 min 28 min

42 min 68 min

3µM

180 min

120 min

60 min20 min0 min

15µM

0 min 12 min 31 min 60 min 120 min

1mM

15 x 15 µm 20 x 20 µm

z-range10 nm

Integrity

of SPB : AFMSupported

planar

bilayer

: DOPC/DPPC (1:1) (25°C)

~ CMC

> CMC

< CMC

DPPC

DOPC

Permeabilization

and remodelization

of LUV: calcein

release, R18

dequenching

and dynamic light scattering

(DLS)

3 µM15 µM25 µM100 µM

)exp1()exp1(% 21 2max

1max

tktkreleased yycalcein CMC

Calcein

release R18 dequenching

Dynamic

light scattering

Permeabilization

and remodelization

of LUV: calcein

release, R18

dequenching

and dynamic light scattering

(DLS)

3 µM15 µM25 µM100 µM

)exp1()exp1(% 21 2max

1max

tktkreleased yycalcein

< CMC

CMC

Permeabilization

and remodelization

of LUV: calcein

release, R18

dequenching

and dynamic light scattering

(DLS)

3 µM15 µM25 µM100 µM

)exp1()exp1(% 21 2max

1max

tktkreleased yycalcein

~ CMC

CMC

Permeabilization

and remodelization

of LUV: calcein

release, R18

dequenching

and dynamic light scattering

(DLS)

3 µM15 µM25 µM100 µM

)exp1()exp1(% 21 2max

1max

tktkreleased yycalcein

Two

release mechanisms

> CMC

CMC

Conclusions : Surfactin

/ membrane interaction

[surfactin] < CMC

[surfactin] ~ CMCSolubilization

of DOPC, pore formation

Erosion of DPPC domains

and transient

defects

Red

= surfactin

[surfactin] >> CMC

Transformation into

micelles, Fusion ?

Conclusions : Surfactin

/ membrane interaction

General conclusions

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

OCH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

HCH2OH

COOH

HO

Membrane raft receptors

(Fas,.) ?

Other

receptor

dependentactivities

General conclusions

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

OCH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

HCH2OH

COOH

HO

Perspectives

Short term-perspectives

Cell

staining

(McIntosh 2008, Garvik

2009)GUV : DOPC:DPPC:DHE (1:1:1)

Biphoton

microscopy

of DHE

Tof-SIMS

SPB (Lorent)Cells(Mazzucchelli

2008)

Perspectives

Long term-perspectives

CH2OH

COOH

O

HO

H

H

HO

HOH

HH

O

OH

H

OH

H

H

H

OCH3

H

O

H

Hemisynthesis

HemolysisActivity

on cancer cells

New anticancer

drugs

?

HN

HN

O

O

NH

HN

OOH

O

O

HN

HNO

O

NH

O

O

O

OH

O

CH2OH

COOH

HO (QSAR)

New nanoparticles