24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Membrane Photo-permeabilization and Importance of the physico-chemical properties of photosensitizers used in PhotoChemical internalization of macromolecules (PCI)
Kerdous R.1, Mojzisova H.1, Brault D.1, Heuvingh J.2, Bonneau S1. 1 FRE
3207 UPMC, 2 UMR 7636 ESPCI
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
• Photosensitizers Definition PDT vs. PCI N H
• PCI Principle Examples A good PCI-PS ?
• Subcellular localisation • Membrane permeabilization Membrane geometry Membrane asymmetry
• Conclusions
N
N H N
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
N
Photosensitizer
N HN
irradiation
Photosensitizer
∆ concentration
N H
Drug activity
Temps
PhotoDynamic Therapy (PDT) Photochemical Internalization (PCI)
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
• Photosensitizers Definition PDT vs. PCI N H
• PCI Principle Examples A good PCI-PS ?
• Subcellular localisation • Membrane permeabilization Membrane geometry Membrane asymmetry
• Conclusions
N
N H N
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Photochemical Internalisation (PCI) Endocytosis of macromolecules et photo-induced release
Proteines DNA … Lysosome Nucleus X
X
X
X X X
X
Photosensitizer
References: Berg et al. Cancer Res.,1999 Berg et al. Oftalmologia, 2003
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Photochemical internalisation of PNAS AlPcS2a-PCI on cell cultures
Photochemical Internalization of a Peptide Nucleic Acid Targeting the Catalytic Subunit of Human Telomerase1
Références: Folini et al. Cancer research, 2003
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Gelonin photochemical internalisation AlPcS2a-PCI In VIVO
Enhanced photodynamic destruction of a transplantable fibrosarcoma using photochemical internalisation of gelonin
Références: Dietze et al. Br. J. Cancer, 2005
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
A good photosensitizer for PCI ? Permeabilization efficiency
Subcellular localization
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
• Photosensitizers Definition PDT vs. PCI N H
• PCI Principle Examples A good PCI-PS ?
• Subcellular localisation • Membrane permeabilization Membrane geometry Membrane asymmetry
• Conclusions
N
N H N
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Disulfonated aluminium phthalocyanine
Charged
Chlorin e6
Three neutralisable charges
Deuteroporphyrin
Two neutralisables charges
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Subcellular localisation 3-sortie
2-flip-flop
1-entrée
-
pH Influence
+
LDL Influence
+ -
transmembrane crossing Références: Bonneau et al. Biochem.Pharmacol, 2004 ; Mojzisova et al. BBA, feb. 2007 and BBA, nov. 2007
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
• Photosensitizers Definition PDT vs. PCI N H
• PCI Principle Examples A good PCI-PS ?
• Subcellular localisation • Membrane permeabilization Membrane geometry Membrane asymmetry
• Conclusions
N
N H N
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Unsatturated lipids in mecanisms of photodynamic processes • Lipid hydroperoxides = important non-radical intermediates of the peroxidative process • LOOH are unstable and decompose to form secondary products such as aldehydes and various breakdown products.
00H
C0=0
C00
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24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Models of lipids membranes Lipidic unilamellar vesicles
• Water permeable, impermeable to solutes
GUV: • Volume fixed by osmolarity • Surface area fixed by the number of lipids • Optical contrast due to the difference of inner and external medium
SUV (TEM)
Used for spectroscopy
5nm
PS
5 à 20 µm
Sucrose NaCl Glucose NaCl
GUV (PCM)
Used for microscopy
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
mTHPC Foscan ®
Chlorin e6
TPCS2a
Φ∆ Three chlorin-macrocycles
Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
Ce6
0,63
TPCS2a
0,66
m-THPC
0,68
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Photo-induced peroxidation Methyl linoleate (ML) → conjugated diene (abs. 234 nm)
In solution Within the membrane
Proportional to Φ∆ Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
m-THPC >TPCS2a>Ce6
DPPC SUV
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
τ= 3ns
τ= 35 µs D~250nm 1O
Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
2
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Photo-induced permeabilization
DOPC-SUV
TPCS2a>Ce6>>m-THPC Photo-oxidation and permeabilization are not proportional Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
11O O22
11 1O O O222
1O
2
Effects induced by charges ? By the depth within the membrane ? Perturbation of the membrane symmetry ? Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
1O
2
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Photo-inducing membrane asymmetry Asymmetrical perturbation of the membrane: PS incubated with GUVs → External leaflet labelled
Ce6
PS added to the lipids during the preparation of the GUVs → Both leaflets labelled
DOPC-GUV
+ Rincing / centrifugation → Inner leaflet labelled
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Chlorin within the inner leaflet
10 µm Speed 7x
Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Shape transitions (Inner leaflet targeted) Pear shape
Prolate vesicle
10 µm
2 seconds / picture
Outward budding Budding Pear shape
1 second / picture
Outward budding 10 µm
Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Chlorin within the external leaflet
10 µm Speed 7x
Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Shape transitions (External leaflet targeted) short time deformation
5 µm
2
11
12
13
Inward budding
14
15
permeabilization
28
45
70 time (s)
short time deformation
5 µm
2
8
9
10
deformation
11
12
Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
13
14
Inward budding permeabilization
15
40
60
80
100 time (s)
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
ADE Model Seifert 1997 Döbereiner 2000
Relative contribution of various events
Shape transitions and spontaneous curvature 100%
Inner leaflet
80% 60%
Both leaflet 40% 20% 0% 20% 40% 60% 80%
100%
Outer leaflet
Outward budding Oblate to prolate transition Long-time fluctuation (>20s)
Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
Short-time fluctuation (0
Oxidation if the inner leaflet
Negative curvature of the photo-sensitized leaflet
∆A C0
int
C ∆Hs
∆H s =
for
A k bT ( σ − σ 0 ) = 4 πr 2 σ 8π k c
2σ C > kc * 0
Lysis tension of 0.15 mN/m if C0* > 42 µm-1 Area decrease of 2.3% in a leaflet Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009
Lysis of the GUV
Area Volume
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
• Photosensitizers Definition PDT vs. PCI N H
• PCI Principle Examples A good PCI-PS ?
• Subcellular localisation • Membrane permeabilization Membrane geometry Membrane asymmetry
• Conclusions
N
N H N
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
Conclusions Depth and asymmetry of photo-modification within the membrane are key of its effect Importance of the localisation of the photosensitizer within the membrane Effticiency of asymmetrically charged PS for PCI Flip-flop, GOVERNED BY the DEPTH Lysosomal localisation + permeabilisation of membranes
Permeabilization Stenching
24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009
OSLO, norvege Department of Radiation Biology
ANBioPhy Biophotonics Group
Kristian Berg group
Rachid Kerdous Halina Mojzisova Daniel Brault
PMMH Julien Heuvingh
CSVB Philippe Maillard
