Kerdous R. 1, Mojzisova H. 1, Brault D. 1, Heuvingh J. 2, Bonneau S 1. FRE 3207 UPMC, 2 UMR 7636 ESPCI

Transcription

1 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 S 1. 1 FRE 3207 UPMC, 2 UMR 7636 ESPCI

2 Photosensitizers Definition PDT vs. PCI PCI Principle Examples A good PCI-PS? Subcellular localisation H H Membrane permeabilization Membrane geometry Membrane asymmetry Conclusions

3 H H 24th Annual Meeting of the G.T.R.V. - Paris, December 7/9, 2009 Photosensitizer irradiation concentration Photosensitizer Drug activity Temps PhotoDynamic Therapy (PDT) Photochemical Internalization (PCI)

4 Photosensitizers Definition PDT vs. PCI PCI Principle Examples A good PCI-PS? Subcellular localisation H H Membrane permeabilization Membrane geometry Membrane asymmetry Conclusions

5 Photochemical Internalisation (PCI) Endocytosis of macromolecules et photo-induced release Proteines DA Lysosome ucleus X X X X X X X Photosensitizer References: Berg et al. Cancer Res.,1999 Berg et al. Oftalmologia, 2003

6 Photochemical internalisation of PAS AlPcS2a-PCI on cell cultures Photochemical Internalization of a Peptide ucleic Acid Targeting the Catalytic Subunit of Human Telomerase1 Références: Folini et al. Cancer research, 2003

7 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

8 A good photosensitizer for PCI? Permeabilization efficiency Subcellular localization

9 Photosensitizers Definition PDT vs. PCI PCI Principle Examples A good PCI-PS? Subcellular localisation H H Membrane permeabilization Membrane geometry Membrane asymmetry Conclusions

10 Disulfonated aluminium phthalocyanine Chlorin e6 Deuteroporphyrin Charged Three neutralisable charges Two neutralisables charges

11 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 and BBA, nov. 2007

12 Photosensitizers Definition PDT vs. PCI PCI Principle Examples A good PCI-PS? Subcellular localisation H H Membrane permeabilization Membrane geometry Membrane asymmetry Conclusions

13 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 C 0 =0 C 0 <0 C 0 >0

14 Afin de vous aider à protéger votre confidentialité, PowerPoint a empêché le téléchargement automatique de cette image externe. Pour la télécharger et l'afficher, cliquez sur Options dans la barre de message, puis cliquez sur Autoriser le contenu externe. 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 Sucrose acl PS Glucose acl 5nm 5 à 20 µm GUV (PCM) Used for microscopy

15 mthpc Foscan Chlorin e6 TPCS2a Φ Three chlorin-macrocycles Ce6 TPCS 2a m-thpc 0,63 0,66 0,68 Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

16 Photo-induced peroxidation Methyl linoleate (ML) conjugated diene (abs. 234 nm) DPPC SUV In solution Within the membrane Proportional to Φ m-thpc >TPCS 2a >Ce6 Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

17 τ= 3ns 1 O 2 τ= 35 µs D~250nm Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

18 Photo-induced permeabilization DOPC-SUV TPCS 2a >Ce6>>m-THPC Photo-oxidation and permeabilization are not proportional Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

19 1 O 2 1 O 2 1 O 2 1 O 2 1 O 2 1 O 2 Effects induced by charges? By the depth within the membrane? Perturbation of the membrane symmetry? Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

20 Photo-inducing membrane asymmetry Asymmetrical perturbation of the membrane: PS incubated with GUVs External leaflet labelled Ce6 DOPC-GUV PS added to the lipids during the preparation of the GUVs Both leaflets labelled + Rincing / centrifugation Inner leaflet labelled

21 Chlorin within the inner leaflet 10 µm Speed 7x Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

22 Shape transitions (Inner leaflet targeted) Prolate vesicle Pear shape 2 seconds / picture Outward budding 10 µm Pear shape Budding 1 second / picture Outward budding Ref.: J. Heuvingh & S. Bonneau; Biophys. J µm

23 Chlorin within the external leaflet 10 µm Speed 7x Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

24 Shape transitions (External leaflet targeted) 5 µm short time deformation Inward budding permeabilization 5 µm short time deformation deformation Inward budding time (s) permeabilization time (s) Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

25 Shape transitions and spontaneous curvature ADE Model Seifert 1997 Döbereiner 2000 Relative contribution n of various events 100% Inner leaflet 80% 60% 40% 20% 0% 20% 40% 60% 80% 100% Outer leaflet Both leaflet Outward budding Oblate to prolate transition Long-time fluctuation (>20s) Short-time fluctuation (<2s) Inward budding Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

26 Shape transitions and spontaneous curvature Effective spontaneous curvature: ext C 0 *= C 0 + απ h a 0 a 0 C>0 int Oxidation if the inner leaflet A C 0 C<0 egative curvature of the photo-sensitized leaflet ext Ref.: J. Heuvingh & S. Bonneau; Biophys. J int Oxidation of the external leaflet

27 Permeabilization of the membrane Ce6 outside Ce6 both (in/out) Ce6 inside Pore diameter between 16 nm to 43 nm [Ce6] Importance of the asymmetry in the permeabilization process Photo-oxidation and permeabilization are not quantitatively correlated Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

28 Permeabilization, curvature and tension Modification of the spontaneous curvature Area Volume Tension of the membrane Lysis of the GUV Hypothesis : the budding induces a tension of 0.15 m/m Ref.: J. Heuvingh & S. Bonneau; Biophys. J. 2009

29 Permeabilization, curvature and tension Modification of the spontaneous curvature Area Volume Tension of the membrane Lysis of the GUV Energy gained when budding a vesicle of radius r H b = 8πk c * ( 1 C r ) 0 Energy necessary to entropically stretch a membrane from a tension σ 0 to a tension σ is: A k bt H s = ( σ σ ) = 8π k c 0 4 πr 2 σ H b > H s for Lysis tension of 0.15 m/m if Ref.: J. Heuvingh & S. Bonneau; Biophys. J C * > 0 2σ k c C 0 * > 42 µm -1 Area decrease of 2.3% in a leaflet Area Volume

30 Photosensitizers Definition PDT vs. PCI PCI Principle Examples A good PCI-PS? Subcellular localisation H H Membrane permeabilization Membrane geometry Membrane asymmetry Conclusions

31 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, GOVERED BY the DEPTH Lysosomal localisation + permeabilisation of membranes Permeabilization Stenching

32 ABioPhy Biophotonics Group Rachid Kerdous Halina Mojzisova Daniel Brault OSLO, norvege Department of Radiation Biology Kristian Berg group PMMH Julien Heuvingh CSVB Philippe Maillard