NIR-Emitting Gold Nanoclusters Encapsulated in PS-b-PEG Polymer Micelles.

Gold nanoclusters (AuNCs) are an emerging type of luminescent probe, featuring good biocompatibility, high photostability, and large Stoke shifts. Their lack of colloidal stability is, however, a drawback for many applications. Here, we report the stabilization of AuNCs emitting in the NIR by a thiol-terminated polystyrene chain (Mn = 5000 g mol-1). The optical properties of this nanocomposite remain invariant for 2 years in THF. To use the PS5k-AuNCs in an aqueous environment, these were encapsulated into polymer micelles using a polystyrene-b-poly(ethylene glycol) copolymer. The resulting hierarchical constructs, with diameters of ca. 125 to 215 nm, have promising properties for applications as luminescent probes such as contrast agents for biomedical imaging.

Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin.

Nucleolin is a multifunctional protein involved in essential biological processes. To precisely localize it and unravel its different roles in cells, fluorescence imaging is a powerful tool, especially super-resolution techniques. Here, we developed polymer-aptamer probes, both small and bright, adapted to direct stochastic optical reconstruction microscopy (dSTORM). Well-defined fluorescent polymer chains bearing fluorophores (AlexaFluor647) and a reactive end group were prepared via RAFT polymerization. The reactive end-group was then used for the oriented conjugation with AS1411, a DNA aptamer that recognizes nucleolin with high affinity. Conjugation via strain-promoted alkyne/azide click chemistry (SPAAC) between dibenzylcyclooctyne-ended fluorescent polymer chains and 3'-azido-functionalized nucleic acids proved to be the most efficient approach. In vitro and in cellulo evaluations demonstrated that selective recognition for nucleolin was retained. Their brightness and small size make these polymer-aptamer probes an appealing alternative to immunofluorescence, especially for super-resolution (10-20 nm) nanoscopy. dSTORM imaging demonstrated the ability of our fluorescent polymer-aptamer probe to provide selective and super-resolved detection of cell surface nucleolin.

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry.

MALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an antioxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any antioxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain-chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pKa of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains.

"Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing αvß3 Integrins.

A new class of "polymultivalent" ligands combining several ligand clusters and a water-soluble biocompatible polymer is introduced. These original conjugates bear two levels of multivalency. They are prepared by covalent coupling of a controlled number of tetrameric cRGD peptide clusters along a well-defined copolymer synthesized by RAFT polymerization. The presence of multiple copies of peptide clusters on the same polymer backbone resulted in a much-higher relative potency than the free cluster reference. Thanks to the "polymultivalency", up to ∼2 orders of magnitude potency enhancement was reached in a competitive cell adhesion assay (nanomolar-range IC50 values). In addition, confocal microscopy and flow cytometry demonstrated that fluorescent "polymultivalent" conjugates (emitting in the far-red/near-infrared region) were able to specifically and selectively label cells expressing αvß3-integrin, the natural receptor of cRGD.

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.

Synthesis of biotinylated α-D-mannoside or N-acetyl ß-D-glucosaminoside decorated gold nanoparticles: study of their biomolecular recognition with Con A and WGA Lectins.

Gold nanoparticles (NPs) functionalized with a mixed shell of well-defined biotinylated glycopolymers and polyethylene glycol (PEG) provide an effective platform for the biomolecular recognition of proteins both in solution and on surfaces. Well-defined biotinylated glycopolymers were first synthesized by the reversible addition-fragmentation chain transfer (RAFT) process. They contain two types of carbohydrate residues either N-acetyl ß-D-glucosaminopyranoside (GlcNAc) or α-D-mannopyranoside (Man) as pendent groups. The biotinylated glycopolymers and polyethylene glycol were subsequently used in the in situ formation of gold glyconanoparticles via an easy photochemical process. The obtained biotinylated glyconanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The bioavailability of the biotin and specific carbohydrate residues at the periphery of the NPs were assessed using the diffraction optic technology (DOT) system. The studies showed the accessibility of the biotin ligands for conjugation to immobilized avidin on the DOTLab biosensor. Furthermore, these avidin conjugated glyconanoparticles were found to selectively immobilize lectins. The specificity of lectin binding was dependent on the type of carbohydrate residues. As such, N-acetyl ß-D-glucosaminoside decorated gold nanoparticles were found to specifically interact with wheat germ agglutinin (WGA) lectin, whereas α-D-mannoside ones were found to specifically interact with Concanavalin A (Con A) lectin.

Schizophrenic behavior of a thermoresponsive double hydrophilic diblock copolymer at the air-water interface.

The thermoresponsive behavior of the rhodamine B end-labeled double hydrophilic block copolymer (DHBC) poly(N,N-dimethylacrylamide)-b-poly(N,N-diethylacrylamide) (RhB-PDMA(207)-b-PDEA(177)) and the 1:1 segmental mixture of PDEA and rhodamine B end-labeled PDMA homopolymers was studied over the range of 10-40 degrees C at the air-water interface. The increase in collapse surface pressure (second plateau regime) of the DHBC with temperature confirms the thermoresponsiveness of PDEA at the interface. The sum of the pi-A isotherms of the two single homopolymers weighted by composition closely follows the pi-A isotherm of the DHBC, suggesting that the behavior of each block of the DHBC is not influenced by the presence of the other block. Langmuir-Blodgett monolayers of DHBC deposited on glass substrates were analyzed by laser scanning confocal fluorescence microscopy (LSCFM), showing schizophrenic behavior: at low temperature, the RhB-PDMA block dominates the inside of bright (core) microdomains, switching to the outside (shell) at temperatures above the lower critical solution temperature (LCST) of PDEA. This core-shell inversion triggered by the temperature increase was not detected in the homopolymer mixture. The present results suggest that both the covalent bond between the two blocks of the DHBC and the tendency of rhodamine B to aggregate play a role in the formation of the bright cores at low temperature whereas PDEA thermoaggregation is responsible for the formation of the dark cores above the LCST of PDEA.

Multiscale fluorescent tracking of immune cells in the liver with a highly biocompatible far-red emitting polymer probe.

The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 µM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 µm thick cleared sections, demonstrated the versatility of the 19K-6H probe.

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology.

We report the site-specific and covalent bioconjugation of fluorescent polymer chains to proteins in live cells using the HaloTag technology. Polymer chains bearing a Halo-ligand precisely located at their α-chain-end were synthesized in a controlled manner owing to the RAFT polymerization process. They were labeled in lateral position by several organic fluorophores such as AlexaFluor 647. The resulting Halo-ligand polymer probe was finally shown to selectively recognize and label HaloTag proteins present at the membrane of live cells using confocal fluorescence microscopy. Such a polymer bioconjugation approach holds great promises for various applications ranging from cell imaging to cell surface functionalization.

Innovative particle standards and long-lived imaging for 2D and 3D dSTORM.

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging.

Exogenous probes with far-red or near-infrared (NIR) two-photon absorption and fluorescence emission are highly desirable for deep tissue imaging while limiting autofluorescence. However, molecular probes exhibiting such properties are often hydrophobic. As an attractive alternative, we synthesized water-soluble polymer probes carrying multiple far-red fluorophores and demonstrated here their potential for live cell and zebrafish embryo imaging. First, at concentrations up to 10 µm, these polymer probes were not cytotoxic. They could efficiently label living HeLa cells, T lymphocytes and neurons at an optimal concentration of 0.5 µm. Moreover, they exhibited a high resistance to photobleaching in usual microscopy conditions. In addition, these polymer probes could be successfully used for in toto labeling and in vivo two-photon microscopy imaging of developing zebrafish embryos, with remarkable properties in terms of biocompatibility, internalization, diffusion, stability and wavelength emission range. The near-infrared two-photon absorption peak at 910 nm is particularly interesting since it does not excite the zebrafish endogenous fluorescence and is likely to enable long-term time-lapse imaging with limited photodamage.

New hydrolyzable pH-responsive cationic polymers for gene delivery: a preliminary study.

Here we want to report the synthesis and the characterization of 2-methylacrylic acid 2-(3-imidazol-1-yl-propionyloxy)ethyl ester (IPEMA), a new methacrylate derivative monomer bearing an hydrolyzable side chain terminated by an imidazole group. The kp/kt(1/2) value for its homopolymerization in N,N-dimethylformamide at 60 degrees C was found to be 0.120 mol(-1/2) x L(1/2) x s(-1/2). The free radical copolymerization of N,N-dimethylaminoethyl methacrylate and this monomer was studied in N,N-dimethylformamide at 60 degrees C, the reactivity ratios of this couple of monomers were determined to be r(DMAEMA) = 1.13 +/- 0.09 and r(IPEMA) = 0.82 +/- 0.09 (using distinct calculation methods). Molecular weights analysis, parallely with refractive index increments measurements, were performed to characterize the obtained polymers. Potentiometric titrations showed the ability of these copolymers to act as a 'proton sponge'. Preliminary study of the copolymers hydrolysis proved that imidazole units could be slowly cleaved from the polymer backbone at 37 degrees C in neutral aqueous buffer. Agarose gel electrophoresis of plasmid DNA/polymer complexes demonstrated the DNA complexing properties of these imidazole-based copolymers.

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).

Fluorescence anisotropy of hydrophobic probes in poly(N-decylacrylamide)-block-poly(N,N-diethylacrylamide) block copolymer aqueous solutions: evidence of premicellar aggregates.

Fluorescent probes, coumarin 153 (C153) and octadecylrhodamine B (ORB), were used to study the self-assembly in water of poly(N-decylacrylamide)-block-poly(N,N-diethylacrylamide), (PDcA(11)-block-PDEA(295); M(n) = 40 300 g mol(-1); M(w)/M(n) = 1.01). From the variation of both the fluorescence intensity and the solvatochromic shifts of C153 with polymer concentration, the critical micelle concentration (CMC) was determined as 1.8 +/- 0.1 microM. On the other hand, steady-state anisotropy measurements showed the presence of premicellar aggregates below the CMC. Time-resolved fluorescence anisotropy evidenced that ORB is located in the premicellar aggregates and the micelle core, while C153 is partitioned between the aggregates and the water phase. The micelle core contains both semicrystalline and amorphous regions. In the semicrystalline regions the probes cannot rotate, while in the amorphous regions the rotational correlation times correlate well with the hydrodynamic volume of the probes. The amorphous region of the micelle core is relatively fluid, reflecting the large free-volume accessible to the probes.

Block copolymer-oligonucleotide conjugates for genotyping on microarrays.

Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.

Preparation of biotinylated glyconanoparticles via a photochemical process and study of their bioconjugation to streptavidin.

We report here the preparation of novel biotinylated glyconanoparticles from well-defined biotinylated glycopolymers and poly(N-isopropylacrylamide) (PNIPAAm) synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization process. The in situ reduction of the biotinylated glycopolymers, PNIPAAm, poly(ethylene glycol), and HAuCl4 via a photochemical process resulted in the formation of biotinylated gold nanoparticles. The multifunctional biotinylated glyconanoparticles were then evaluated for their bioconjugation toward streptavidin using UV-vis spectroscopy and surface plasmon resonance (SPR). The biotinylated nanoparticles underwent aggregation in the presence of streptavidin as revealed by spectrophotometry, which indicates the accessibility of the biotin for conjugation. These results were further confirmed by surface plasmon resonance even in the case of surface-immobilized streptavidin.

Versatile precursors of functional RAFT agents. Application to the synthesis of bio-related end-functionalized polymers.

Biomolecule-polymer conjugates are widely used in bio-related fields, but their synthesis is often tricky, especially the introduction of a single biomolecule at one chain end. This paper describes a new straightforward approach to prepare such conjugates via RAFT polymerization. By designing appropriate bio-related RAFT agents, polymer chains of controlled chain length (Mn = 10 000-40 000 and PDI < 1.1) carrying a single biomolecule as an alpha-end group (a sugar or a biotin) linked by a stable amide bond are obtained. Considering the versatility of the RAFT process, this strategy appears to be very attractive for the design of a variety of conjugates.

Water-soluble dendrigrafts bearing saccharidic moieties: elaboration and application to enzyme linked OligoSorbent Assay (ELOSA) diagnostic tests.

The synthesis of a series of water-soluble galactopyranose-functionalized polystyrene-polyvinyl ether dendrigrafts and their characterization (in solution and thin solid deposits) have been achieved. The presence of external galactopyranose groups on dendritic polymers has been exploited to prepare dendrigraft-oligonucleotide conjugates using a simple one-step coupling procedure with amino-ended oligonucleotides (ODNs). Several parameters such as the peripherical density of hydrophilic branches, the polymerization degree of polystyrene or poly(hydroxyethyl vinyl ether) blocks, and the number of galactopyranose groups were tuned. A capture test with short labeled complementary ODNs (25 bases) confirmed the presence of covalently bound ODNs on various kinds of dendrigrafts. The ability of the dendritic polymers to enhance the sensitivity of enzyme-linked oligosorbent assay (ELOSA) diagnostic tests (detection of hepatitis B virus, DNA target of 2400 bases) was then evaluated, especially the influence of the macromolecular architecture and the impact of the structural parameters. The dendrigraft-ODN conjugate with the lower saccharide external density was found to lead to a very significant amplification of the fluorescence signal, corresponding to a limit of sensitivity of 10(9) DNA copies per milliliter (instead of 10(11) DNA copies per milliliter without using dendrigrafts). Conversely, the dendrigrafts exhibiting a very high number of branches and galactopyranose groups at their periphery were not able to induce a better sensitivity due to steric hindrance generated by the peripheral congestion on these polymers.

Thin films of hydrophobically modified poly(N,N-dimethylacrylamide).

The behavior of a poly(N,N-dimethylacrylamide) hydrophobically modified by incorporating 0.33 mol % of a pyrenyl derivative, [4-(1-pyrenyl)butyl]amine hydrochloride (PY) and 3.56 mol % of dodecylamine (DO) has been studied at the air/water interface. Surface pressure-area isotherm measurements show that the film is initially anchored by the hydrophobic groups at the air-water interface with a pancake-like structure and, with increasing surface pressure, evolves to a quasi mushroom structure, finally reaching a brush configuration at high pressures. Monolayers of this polymer were transferred to silica substrates using the Langmuir-Blodgett (LB) technique at 5, 15, and 25 mN.m(-1). The properties of the LB films were studied by steady-state and time-resolved fluorescence as well as by atomic force microscopy. The results show that the aggregates formed at low pressures are disrupted by pressure increase, while the water-soluble poly(N,N-dimethylacrylamide) becomes dissolved in the water subphase.