Role of plasma LDL and tumoral microenvironment in tetrapyrrole-photosensitizers cellular uptake: a physico-chemical approach
Stéphanie Bonneau, Halina Mojzisova, Christine Vever-Bizet, Daniel Brault
BIOMOCETI Laboratoire de Biophysique Moléculaire, Cellulaire et Tissulaire Université Paris 6 / CNRS UMR 7033 12th ESP, Bath, sept. 2007
P. & M. Curie Univ., Paris
INTRODUCTION
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Mecanisms of tumoral destruction • Two selectivity factors Restriction of the light irradiation to deased aera Differential retention by the compartments
• Vascular damages Importance of the injection-irradiation delay and of the pharmacokinetics
• Immune effects • Cellular death (apoptosis or necrosis) Importance of the sub-cellular localisation 12th ESP, Bath, sept. 2007
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Pharmacokinetics and PDT • Importance of the dynamic aspects in PDT ∆ concentration
irradiation
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Photosensitizer Conventionnal drug
time
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Photosensitizers transport in organism Variability + modification as a function of dose and vectorisation mode Intravenous injection
Intestinal absorption
Endogenous Production
Hepatic metabolism Transmembrane crossing (diffusion or endocytosis)
Systemic circulation Protein association Solubility
Hepatic metabolism
Hepatic elimination 12th ESP, Bath, sept. 2007
Target
Renal elimination
Local properties of the circulation P. & M. Curie Univ., Paris
MECANISMS OF THE SPECIFIC RETENTION IN TUMORAL CELLS
AlPcS2a
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Ce6
DP
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Fixation of PS to LDL PF = A0-A1.exp(-k1t)-A2.exp(-k2t)
{
où K1, 2 = ½ ∑k±[ (∑k)2- 4(kdP.kdL+k’aL.kdP+k’aP.kdL)]
}
½
k = (kaP +kaL).[LDL]+kd association
PF = A0-A1.exp(-k1t)-A2.exp(-k2t) k1 = kdP et k2 = kdL dissociation 12th ESP, Bath, sept. 2007
Bonneau and al. 2002 Mojzisova and al. 2007 Bonneau and al. 2007
P. & M. Curie Univ., Paris
Fixation of PS to LDL • Two fixation types
« Classe P » → lipids/apoprotein interface « Classe L » → incorporation in the lipids
Limited binding to the Apo → recognition by the « B-E » receptors
• High affinity
MODIFIED BY
pH
LDL = good carriers for photosensitisers Role of micro-environment
• Relative rapidity of events Association → ≈ 10-4 s Dissociation → ≈ 0.1 s Possibility of exchanges between the potential carriers 12th ESP, Bath, sept. 2007
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Membrane models
• Dioleoylphosphatidylcholine (C18:1) • Extrusion → Unilamellar vesicules
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Signal of fluo.
The PS-membrane interaction
0
kon
kin
koff
kex
0.05 time (s)
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ESP, Bath, sept. 2007
0.1
0
1 time (s)
2
DP P. & M. Curie Univ., Paris
Membrane association mecanisms and protonation forms COOH
COOH
H H33C C H
5.3 ?
NNH NN H H NN HNN
pKN1 = 4,4 H H33CC
NH
pKN2 = 2,7
H
N
NH +HN
HCH3 CH3
pKA1 = 5,4
COOH
NH
(Dimer forms) COOH
N
Entrance disfavoured by the dimer forms
+
COOH
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N
pKA2 = 6,0
HN
Flip-flop favoured COOH
NH HN
COOH
6.9 et 7.1
3
COOH
+
NH HN
COOH
CH3 H CH
Exit facilitated by the carboxylic charges
COO
NH N
COO
N HN
COO
P. & M. Curie Univ., Paris
≈ 5.3
pKN1
Efficiancy window
pKA1
≈7
pKA2
flip-flop basic pH
acidic pH association
Nitrogen charged
Nitrogens neutralisation
Charge of the carboxylic chains Not deep incorporation
association to the membrane
dimerisation Entrance
Facilitated Exit
Kuzelova K and al. 1995 Bonneau and al. 2004 12th ESP, Bath, sept. 2007
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Behaviors towards the membranes 3-exit 2-flip-flop
DP 1-entrance
AlPcS2
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Ce6
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Cellular incorporation of DP and AlPcS2 Dye solution AlPc, DP or Ce6
Dye pre-loaded on LDL LDL-AlPc, LDL-DP or LDL-Ce6 or
control Without dye
or
Incubation of the cells with one of the solutions 15 min, 5% CO2
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Cellular incorporation of Ce6
• Influence of pH • Implication of LDL 12th ESP, Bath, sept. 2007
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CELLULAR PATHWAY OF THE PS-INTERNALISATION
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Cellular internalisation pathway and subcellular localisation Endosome
Lysosome
Endocytosis vesicle pH≈6
Endocytosis bulk or LDL-mediated
pH≈5
Nucleus
pH≈7.4
Passive diffusion
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Golgi apparatus Plasma membrane Endoplasmic Mitochondria Reticulum P. & M. Curie Univ., Paris
Cells incubation with the porphyrin
DP
15 min incubation
DP + LDL
• Membranous and cytosolic labeling • No modification of the localisation by the LDL Fast relocalisation between the different lipidic systems 12th ESP, Bath, sept. 2007
P. & M. Curie Univ., Paris
Cells incubation with the disulfonated aluminium phthalocyanine 15 min incubation
AlPcS2 AlPcS2
AlPcS2 + LDL Lyso-tracker green
Colocalisation
• Punctual localised labeling, no redistribution in the various membranes The endocytosis pathway governs the subcellular localisation 12th ESP, Bath, sept. 2007
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Cells incubation with the chlorin e6
Ce6
15 min incubation
Ce6 + LDL
• Labeling of the endocytosis vesicles and lysosomes • Weak membranous labeling The punctual localisation is enhanced by the LDL preloading 12th ESP, Bath, sept. 2007
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Mecanisms of the cellular internalisation
• LDL systemic carriers • Influence on internalisation process governed by: - rate of transmembrane crossing - exchanges inter-carriers 12th ESP, Bath, sept. 2007
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Acknowledgments Physico-chemistry Cellular Biophysics Spectroscopies
Cellular Biology
UMR 7033 – Biophotonic team Daniel Brault Christine Vever-Bizet Kahina Tarhouni Frank Surreau Halina Mojzisova Geneviève Bourg-Heckely INSERM Patrice Morlière Jean Claude Mazière
Microscopic experiments
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Muséum National d’Histoire Naturelle Marc Gèze P. & M. Curie Univ., Paris
