Photodynamic therapy: Use of poly(ethyleneoxide-b--caprolactone) nanoparticles to deliver pheophorbide a

http://imrcp.ups-tlse.fr

Ehrhart Jrme1, Mingotaud Anne-Franoise1, Mingotaud Christophe1, Vicendo Patricia1, Violleau Frdric2, Souchard Jean-Pierre1, Luc Wasungu3, Teissi Justin 3, Rols Marie-Pierre3, Bonneau Stphanie 4, Kerdous Rachid 4, Sureau Franck 4 1Laboratoire des IMRCP, CNRS, UMR 5623, Universit de Toulouse, 118 route de Narbonne, 31062 Toulouse Cedex, France 2 Ecole dIngnieurs de Purpan, Lab. dAgro-Physiologie, UPSP/DGER 115, 75 voie du TOEC, BP 57611, 31076 Toulouse cedex 03, France 3 Laboratoire de Pharmacologie et Biologie Structurale, CNRS, UMR 5089, 205 Route de Narbonne, 31077 Toulouse 4 Lab. AnBioPhys, universit Paris 6, 5 rue Henri Desbrures, Gnopole campus 1, 91030 Evry, France

Photodynamic therapy
Photosensitizer (PhotoS) O2 ROS ROS Tissue destruction by photo-oxidation
PS 1-2 mg/Kg

hn 1- Detection
1-2 days IV route

Shortcomings
Oesophagus cancer Before treatment

ROS

O2 O2 Tumor O2

Light (Lamp, laser)

2- Destruction Oesophagus cancer 6 months after treatment Photofrin 2 mg/kg IV 630nm, 200 J/linear cm

Treated tissue should be thin Selectivity of PhotoS not always optimal PhotoS poorly soluble in water, formation of aggregates, leading to a strong decrease in efficiency

(Laser, microcamera)

Targeting of PhotoS

Passive targeting : characterization of PEO-PCL self-assemblies incorporating Pheophorbide a

O MeO O n O m H

Photocytotoxicity and cell penetration

Photocytotoxicity (MCF7, test MTT)
100 60 40 20 0 10-8
2,5 5 7,5 Pheo/micelle 80 CI50 4,1x10-8M Pheo CI50 10,2x10-8M

Intracellular fluorescence (ua, 107 cells MCF7, 3h)

12000 10000 8000 6000 4000

PEO-PCL n 45, m 23 Mn PEO = 2000 Mn PCL = 2600

Self-assembly In water (with or without Pheo)

10-7

2,5

5,0

C (mol.L-1)

10-6

2000

DLS

TEM

AFM
Scattered intensity (cm )

SANS

Pheo/micelle more photocytotoxic Better cell penetration

Pheo/micelle Pheo 2,5x10-7 M 2,5x10-7 M

Fluorescence microscopy (HCT116 cells)

Pheo 37C 4C 37C NanoPheo 4C

-1

10

500 nm 100 nm

0.1 0.01 0.1 q ( )

-1

d= 20nm 3, hairy sphere Asymmetrical Flow Field-Flow Fractionation (AsFFFF-[MALS-QELS-UV])

Dh (qels) nm 20.2 Dh AsFFFF nm 19.5 nagg Ip

Penetration kinetics Penetration kinetics by flow cytometry by fluorescence microscopy (HCT116 cells, [Pheo]=10-6M, (MCF7 cells, [Pheo]/[polymer]=1/50, PEO-PCL 2000-2600) PEO-PCL 5000-4000)
500 400 300 200 100 0
NP 1/50 37C NP 1/50 4C NP 1/30 37C NP 1/30 4C Pheo 37C Pheo 4C

Fluorescence intensity

3000

AsFFFF analysis in correlation with TEM, AFM and DLS Monodisperse sample

Fluorescence intensity

2500 2000 1500 1000 500 0 0

NP Pho 1e-6M Pho 1e-6M NP Pho 1e-7M Pho 1e-7M

367

1.013

Fractograms with increasing quantity of Pheo

UV signal integration 0,25 UV Signal 0,2 0,15 0,1 0,05 0 0 5 -1/50-1/30-1/20-1/10-1/5-1/210

Determination of the maximum loading capacity

0,4 0,3 0,2 0,1 0 0 0,1 0,2 0,3 0,4 0,5 0,6 ratio npheo/npolymer

20

40

60 80 100 120 140 time (min)

Cell penetration mainly by endocytosis Better and faster cell penetration confirmed No strong difference between MCF7 and HCT116 for NP penetration

2 3 Time (h)

Conclusion
Self-assembly of PEO-PCL polymer : 20 nm Nanoparticles Maximum loading capacity at 1/10 ratio (mol/mol) for PEO-PCL 2000-2600 Phototoxicity 2.5 times higher with NPs Better and faster cell penetration of encapsulated Pheo Penetration of encapsulated Pheo mainly by endocytosis perinuclear localization either on MCF7 or HCT116 cells
"Monomeric Pheophorbide(a)-containing Poly(ethyleneglycol-b-e-caprolactone) micelles for photodynamic therapy , Knop K., Mingotaud A.-F., El-Akra N., Violleau F., Souchard J.-P., Photochem. Photobiol. Sci. 8(3) (2009) 396-404 Asymmetrical flow field-flow fractionation with multi-angle light scattering and quasi elastic light scattering for characterization of poly(ethyleneglycol-b-e-caprolactone) block copolymer self-assemblies used as drug carriers for photodynamic therapy Ehrhart J., Mingotaud A.-F., Violleau F. J. Chrom. A (2011) under press

Pheo incorporated into the micelle No size change when loading with Pheo Maximum loading capacity : ratio 1/10