Arnaud Favier

Nanocarriers with ultrahigh chromophore loading for fluorescence bio-imaging and photodynamic therapy

We describe the design of original nanocarriers that allows for ultrahigh chromophore loading while maintaining the photo-activity of each individual molecule. They consist in shells of charged biocompatible polymers grafted on gold nanospheres. The self-organization of extended polymer chains results from repulsive charges and steric interactions that are optimized by tuning the surface curvature of nanoparticles. This type of nano-scaffolds can be used as light-activated theranostic agents for fluorescence imaging and photodynamic therapy. We demonstrate that, labeled with a fluorescent photosensitizer, it can localize therapeutic molecules before triggering the cell death of B16-F10 melanoma with an efficiency that is similar to the efficiency of the polymer conjugate alone, and with the advantage of extremely high local loading of photosensitizers (object concentration in the picomolar range).

Biocompatible well-defined chromophore–polymer conjugates for photodynamic therapy and two-photon imaging

A versatile approach is introduced for the synthesis of well-defined, biocompatible conjugates combining two-photon chromophores and hydrophilic multifunctional polymers synthesized by RAFT controlled radical polymerization. As an illustration, two different classes of conjugates carrying multiple fluorophores (based on an anthracene moiety, Anth) or photosensitizers (based on a dibromobenzene moiety, DBB) along the polymer chain were elaborated for bioimaging and photodynamic therapy (PDT) applications, respectively. In both cases, the polymer greatly improved the solubility in biorelevant media as well as the cell uptake. Anth conjugates provided high quality fluorescence microscopy images using both one- and two-photon excitation. DBB conjugates potently induced the death of cancer cells upon photoactivation.

Biotin-end-functionalized highly fluorescent water-soluble polymers

Many biosensing and imaging systems use fluorescence detection. We present the synthesis of biotin-end-functionalized highly fluorescent water-soluble polymers for potential use in biotin–avidin systems. Statistical polymers of N-acryloylmorpholine (NAM) and N-acryloxysuccinimide (NAS) were prepared by RAFT polymerization using a biotinylated chain transfer agent that ensured 95% end-functionalization of the chains. They were further labeled with a lucifer yellow (LY) dye, yielding 7 to 62 LY fluorophores per polymer chain. The resulting polymers exhibited reduced fluorescence self-quenching, with 7- to 43-fold higher brightness than free LY dye. In addition, they featured low pH sensitivity and very good photobleaching resistance. Moreover, we showed that a more extended polymer conformation was beneficial to the binding of the terminal biotin with streptavidin. This work paves the way for the development of polymers for signal amplification in biosensing assays, labeling of biotin-receptors at cell surfaces in some cancer studies, labeling of antibodies and microscopy imaging purposes.

Plasmonic bipyramids for fluorescence enhancement and protection against photobleaching

A great number of studies focus their interest on the photophysical properties of fluorescent hybrid gold nanoparticles for potential applications in biotechnologies such as imaging and/or treatment. Spherical gold nanoparticles are known to quench a chromophore fluorescent signal, when moieties are located in their close vicinity. The use of a polymer spacer on such a system allowed only partial recovery of the dye emission by controlling the surface to dye distance. Gold-based anisotropic sharp nanostructures appear to exhibit more interesting features due to the strong electric field generated at their edges and tips. In this paper, a complete study of hybrid fluorescent bipyramidal-like gold nanostructures is presented. We describe the chemical synthesis of gold bipyramids functionalized with fluorescent water-soluble polymers and their photophysics both in solution and on a single object. We show that the use of a bipyramidal shape instead of a spherical one leads to total recovery of the fluorescence and even to an enhancement of the emission of the dyes by a factor of 1.4.

Synthesis of multifunctional lipid–polymer conjugates: application to the elaboration of bright far-red fluorescent lipid probes

A new class of lipid-ended polymer conjugates presenting reactive sites regularly distributed along the polymer chain were synthesized using RAFT polymerization. The chosen modular approach enables preparation of different lipid families by tuning the nature of the phospholipid α-end, the molecular weight and the lateral functions of the polymer chain. The multiple activated ester functions of the conjugates can indeed be used for the efficient coupling of a great variety of amino-containing entities of interest. In this study, we elaborated original fluorescent lipid–polymer probes for optical microscopy by coupling along the chain a controlled number of chromophores emitting in the far-red where auto-fluorescence and light absorption by biological samples are limited. Water-soluble fluorescent lipid probes exhibiting an enhanced brightness were obtained. As a proof of concept, these probes were able to efficiently label the lipid bilayer of liposomes of various sizes. Such multifunctional lipid-ended polymers thus exhibit great potential to functionalize model and natural lipid assemblies.

Water-soluble chromophores with star-shaped oligomeric arms: synthesis, spectroscopic studies and first results in bio-imaging and cell death induction

A simple polymerisation strategy allows water solubilisation of chromophores for biophotonics, with good conservation of their fluorescence quantum-yield. Preliminary investigations show that the resulting objects are valuable candidates for photodynamic therapy and two-photon fluorescence imaging.

Two-Photon Photosensitizer–Polymer Conjugates for Combined Cancer Cell Death Induction and Two-Photon Fluorescence Imaging: Structure/Photodynamic Therapy Efficiency Relationship

One of the challenges of photodynamic therapy is to increase the penetration depth of light irradiation in the tumor tissues. Although two-photon excitation strategies have been developed, the two-photon absorption cross sections of clinically used photosensitizers are generally low (below 300 GM). Besides, photosensitizers with high cross section values are often non-water-soluble. In this research work, a whole family of photosensitizer-polymer conjugates was synthesized via the covalent binding of a photosensitizer with a relatively high cross section along a biocompatible copolymer chain. The resulting photosensitizer-polymer conjugates were water-soluble and could be imaged in cellulo by two-photon microscopy thanks to their high two-photon absorption cross sections (up to 2600 GM in water, in the NIR range). In order to explore the structure/photodynamic activity relationship of such macromolecular photosensitizers, the influence of the polymer size, photosensitizer density, and presence of charges along the polymer backbone was investigated (neutral, anionic, cationic, and zwitterionic conjugates were compared). The macromolecular photosensitizers were not cytotoxic in the absence of light irradiation. Their kinetics of cellular uptake in the B16-F10 melanoma cell line were followed by flow cytometry over 24 h. The efficiency of cell death upon photoactivation was found to be highly correlated to the cellular uptake in turn correlated to the global charge of the macromolecular photosensitizer which appeared as the determining structural parameter.

Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins

Site-specific labeling of proteins is a valuable tool to study biological processes. We show that it can be achieved using fluorescent polymer probes synthesized by RAFT polymerization. Thanks to an original control agent functionalized with a nitrilotriacetic acid (NTA) ligand, an α-NTA polymer probe was prepared to selectively label histidine-tagged recombinant proteins.

Fluorescent gold nanoparticles with chain-end grafted RAFT copolymers: influence of the polymer molecular weight and type of chromophore

Fluorescent gold nanoparticles (GNPs) were prepared by chain-end grafting of RAFT copolymers bearing multiple fluorophores distributed along the chain. Two different synthetic approaches in water were first studied with well-defined and biocompatible homopolymers. Both led to very stable samples and the corona thickness at the gold surface increased with the polymer molecular weight. The two-step ligand exchange approach was then selected to synthesize GNPs grafted with polymer–chromophore conjugates exhibiting different molecular weights, backbone architectures and chromophores. Again, the thickness of the organic corona increased with the polymer molecular weight but was also dependent on the conformation of the conjugate chains which depends on the nature of the chromophores. By adjusting these parameters, it was possible to control the average chromophore–gold distance which is of paramount importance for the fluorescence properties of the nanoparticles. We indeed showed that the fluorescence of the hybrid nano-objects increased with the corona thickness.

Labeling of native proteins with fluorescent RAFT polymer probes: application to the detection of a cell surface protein using flow cytometry

Conjugation of fluorescent polymer chains synthesized by RAFT controlled radical polymerization with native proteins was achieved by introducing an activated ester function at their ω-chain-end via a very efficient thiol–ene click post-polymerization functionalization strategy. These fluorescent polymer probes covalently reacted with the lysine amino groups of native proteins to form bright protein–polymer fluorescent conjugates. In particular, streptavidin was labeled with various polymer probes and it was shown that it retained in each case its ability to selectively bind biotinylated compounds. Finally, streptavidin labeled with fluorescent polymer chains was used to reveal biotinylated antibodies, enabling the detection of CD40 receptors at the surface of live dendritic cells.