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International audience; A key principle of nanomedicine is to use nanoparticles (NPs) to improve the transport of active and/or contrast agents to a target tissue and to strengthen intra-tissue accumulation. Several nanoconstructions have been investigated regarding anticancer drug delivery, most of them based on lipid or polymer structures (Marchal et al. 2015). Similar to chemotherapeutic agents, the use of biocompatible nanoscale systems for photosensitizers (PSs) delivery in photodynamic therapy (PDT) of cancer demonstrated a significant improvement in PSs bio-availability, -distribution and efficacy of the treatment (Marchal et al., 2015; Abrahamse et al. 2016). PDT is based on the combined action of PS, light and molecular oxygen, leading to the generation of toxic reactive oxygen species (ROS) with the resulting damage of malignant and nonmalignant cells alike, depending on PS distribution (Agostinis et al. 2011; Castano et al., 2004). Vectors allowing both to avoid the fast release of relevant molecules and their high local concentration have emerged in recent years, these are dendrimers. Dendrimers are regular hyperbranched macromolecules a few nanometers in diameter (2-10 nm) and which may have on their surface different functional groups. Because of their low cytotoxicity and their solubility in water polyamidoamines or “PAMAM”-dendrimers appear as a very attractive class of biocompatible nanovectors for drug targeting in cells and tissues (Lee et al. 2005). Thanks to the many functional groups on the periphery of the dendrimer, it is easy to graft therapeutic molecules such as photosensitizers, stealth elements (polyethylene glycol), or targeting moieties (aptamers, antibodies, etc...) while controlling the size and lipophilicity of the carrier. All this will allow monitoring and optimizing cellular uptake, biodistribution and pharmacokinetics of the molecule of interest. The present study investigates generation 4.5 PAMAM dendrimer, covalently functionalized with the PS Chlorin e6 (Ce6). The singlet oxygen generation efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. This construction allows the vectorization of 32 Ce6 molecules per dendrimer. In vitro, PAMAM dendrimers improve the PDT efficiency of Ce6 by promoting their cellular internalization via an active endocytosis mechanism. However, the PDT efficiency of NPs is limited by the high local concentration of Ce6 at the periphery of dendrimers decreasing production of singlet oxygen. Ce6 release could restore Ce6 photophysical properties and as such improve the PDT efficiency of NP. Thus, the next step of this work was to design a cleavable NP able to release the Ce6 under esterase activity. In solution the NP characterization demonstrated that the photophysical properties of Ce6 were recovered after their release from the NP. This cleavable construction displays promising perspectives for future PDT applications.

Photodynamic therapy (PDT) is a modality of cancer treatment, involving a combined action of molecular oxygen, light and photosensitizers (PS). However, the PSs suffer from a low solubility in aqueous media and limited tumor accumulation, diminishing the treatment success. Presently, particular attention is paied to the development of dendrimer-based nanoparticles (NP) that are able to overcome the shortcomings of the PSs. The present study investigates the poly(amidoamine) dendrimer (PAMAM), a tridimensional polymeric macromolecule, covalently functionalized with the PS Chlorin e6 (Ce6). The singlet oxygen generation efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. This construction allows the vectorization of 32 Ce6 molecules per dendrimer. In vitro, PAMAM dendrimers improve the PDT efficiency of Ce6 by promoting their cellular internalization via an active endocytosis mechanism. However, the PDT efficiency of NPs is limited by the high local concentration of Ce6 at the periphery of dendrimers decreasing production of singlet oxygen. Ce6 release could restore Ce6 photophysical properties and as such improve the PDT efficiency of NP. Thus, the next step of this work was to design a cleavable NP able to release the Ce6 under esterase activity. In solution the NP characterization demonstrated that the photophysical properties of Ce6 were recovered after their release from the NP. This cleavable construction displays promising perspectives for future PDT applications; La thérapie photodynamique (PDT) est une modalité de traitement des cancers prometteuse, mettant en jeu une action combinée de l’oxygène moléculaire, de la lumière et d’un photosensibilisateur (PS). Néanmoins, les PSs utilisés souffrent d’une faible solubilité dans les milieux aqueux ainsi que d’un tumorotropisme limité qui sont des barrières à la réussite du traitement. Ainsi, actuellement, une attention particulière est portée au développement de nanoparticules (NPs) capables de pallier les défauts des PSs. Notre travail a consisté à étudier des dendrimères poly(amidoamine) (PAMAM), macromolécules polymériques tridimensionnelles, conjugués via une liaison covalente au PS, la Chlorine e6 (Ce6). Cette construction nous a permis de vectoriser 32 molécules de Ce6 par dendrimère. La production d’oxygène singulet et l’émission de fluorescence ont été modérément affectées par le greffage covalent de la Ce6 aux NPs. In vitro, les dendrimères PAMAM ont permis d’accroitre l’efficacité PDT de la Ce6 en potentialisant son internalisation cellulaire via un mécanisme actif d’endocytose. Néanmoins, l’efficacité PDT des NPs est limitée par la concentration locale élevée en Ce6 en périphérie des dendrimères qui réduit son rendement quantique en oxygène singulet moléculaire, espèce cytotoxique. Une libération de la Ce6 permettrait ainsi de potentialiser l’efficacité PDT des NPs en restaurant notamment les propriétés photophysiques de la Ce6. La suite de ce travail a été de concevoir une NP capable de libérer la Ce6 sous l’action d’estérases retrouvées dans les cellules. Leur caractérisation a permis de démontrer en solution que les propriétés photophysiques de la Ce6 étaient rétablies à la suite de son relargage des NPs. Cette dernière construction clivable est prometteuse pour de futures applications en PDT

International audience; Context: The photosensitizers vectorization enables better tumor localization and therefore a better tumoricidal PDT efficacy. Dendrimers are hyperbranched macromolecules that are considered as an attractive class of biocompatible nanovectors for drug targeting in tissues. In this study we report a novel PDT drug-carrier system, composed of chlorin e6 grafted to the periphery groups of poly(amido amine) dendrimers (PAMAM). Materials & Methods: The size and zeta potential of our nanoparticles were determined by dynamic light scattering (DLS). The nanoparticle singlet oxygen generation in pharynx carcinoma (FaDu) cells was assessed by luminescence at 1268 nm. Phototoxicity was evaluated by MTT assay. Uptake was assessed by chemical extraction associated with fluorescence spectroscopy measurements. Finally, the uptake mechanism was determined by the use of endocytosis specific inhibitors. Results: Ce6-dendrimers nanoparticles displayed a 40 times greater uptake along with a 25 fold higher photocytotoxic activity in FaDu cells compared to free Ce6. Also, a much longer singlet oxygen lifetime was demonstrated for Ce6-dendrimers compared to Ce6. Cellular incorporation of nanoparticles was completely inhibited at 4°C, demonstrating that uptake was mediated via endocytosis. Further investigations using specific inhibitors revealed that incorporation was partially mediated by macropinocytosis pathway. Conclusion: Dendrimeric based nanoparticles showed a good photodynamic efficacy towards cancer cells in vitro, suggesting them as a promising vehicle for PDT. In the future we shall test dendrimer-based nanoparticles in vivo, in pre-clinical models.