Ludovic Richert

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an “in” and “out” diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Improving biocompatibility of implantable metals by nanoscale modification of surfaces: an overview of strategies, fabrication methods, and challenges.

The human body is an intricate biochemical-mechanical system, with an exceedingly precise hierarchical organization in which all components work together in harmony across a wide range of dimensions. Many fundamental biological processes take place at surfaces and interfaces (e.g., cell-matrix interactions), and these occur on the nanoscale. For this reason, current health-related research is actively following a biomimetic approach in learning how to create new biocompatible materials with nanostructured features. The ultimate aim is to reproduce and enhance the natural nanoscale elements present in the human body and to thereby develop new materials with improved biological activities. Progress in this area requires a multidisciplinary effort at the interface of biology, physics, and chemistry. In this Review, the major techniques that have been adopted to yield novel nanostructured versions of familiar biomaterials, focusing particularly on metals, are presented and the way in which nanometric surface cues can beneficially guide biological processes, exerting influence on cellular behavior, is illustrated.

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Analysis of the supramolecular organization of enzymes in the lignin pathway shows that cytochrome P450s oligomerize and move along with the very mobile plant endoplasmic reticulum. Their expression favors relocalization of their soluble partner proteins nearer the membrane and association of sequential enzymes in the pathway. Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.

Virus‐Sized DNA Nanoparticles for Gene Delivery Based on Micelles of Cationic Calixarenes

Macrocyclic amphiphilic molecules based on calix[4]arenes are highly attractive for controlled supramolecular assembly of DNA into small nanoparticles, since they present a unique conical architecture and can bear multiple charged groups. In the present work, we synthesized new amphiphilic calixarenes bearing cationic groups at the upper rim and alkyl chains at the lower rim. Their self-assembly in aqueous solution was characterized by fluorescent probes, fluorescence correlation spectroscopy, dynamic light scattering, gel electrophoresis and atomic force microscopy. We found that calixarenes bearing long alkyl chains (octyl) self-assemble into micelles of 6 nm diameter at low critical micellar concentration and present the unique ability to condense DNA into small nanoparticles of about 50 nm diameter. In contrast, the short-chain (propyl) analogues that cannot form micelles at low concentrations failed to condense DNA, giving large polydisperse DNA complexes. Thus, formation of small DNA nanoparticles is hierarchical, requiring assembly of calixarenes into micellar building blocks that further co-assemble with DNA into small virus-sized particles. The latter showed much better gene transfection efficiency in cell cultures relative to the large DNA complexes with the short-chain analogues, which indicates that gene delivery of calixarene/DNA complexes depends strongly on their structure. Moreover, all cationic calixarenes studied showed low cytotoxicity. Thus, this work presents a two-step hierarchical assembly of small DNA nanoparticles for gene delivery based on amphiphilic cone-shaped cationic calixarenes.

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Youngs modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles

The current challenge in the field of fluorescent nanoparticles (NPs) for bioimaging is to achieve extreme brightness and external control of their emission using biodegradable materials. Here we propose a new concept of fluorescent polymer NPs, doped with ionic liquid-like salts of a cationic dye (octadecyl rhodamine B) with a bulky hydrophobic counterion (fluorinated tetraphenylborate) that serves as spacer minimizing dye aggregation and self-quenching. The obtained 40-nm poly(D,L-lactide-co-glycolide) NPs containing up to 500 dyes are brighter than quantum dots and exhibit photo-induced reversible on/off fluorescence switching, never reported for dye-doped NPs. We show that this collective switching of hundreds of dyes is due to ultrafast excitation energy transfer and can be used for super-resolution imaging. These NPs, being spontaneously endocytosed by living cells, feature high signal-to-noise ratio and absence of toxicity. The counterion-based concept opens the way to a new class of nanomaterials for sensing, imaging and light harvesting.

Use of dopamine polymerisation to produce free-standing membranes from (PLL-HA)n exponentially growing multilayer films

In this communication, we demonstrate that dopamine is able to undergo a polymerisation process in (PLL-HA)n polyelectrolyte multilayer films, and that this polymerisation is of the same nature as in solution at pH 8.5. This polymerisation changes the chemical composition and decreases the mobility of the PLL chains in the film, and ultimately allows the easy detachment of the film as free-standing membranes with 0.1 M HCl solutions.

Dipolar 3-methoxychromones as bright and highly solvatochromic fluorescent dyes

Herein, three environment-sensitive (solvatochromic) fluorescent dyes presenting a strong electron acceptor 3-methoxychromone unit and varied electron donor 2-aryl were developed. All three dyes showed remarkable polarity-dependent shifts of the emission maximum, which increase with extension of the dye conjugation. For the 3-methoxychromone bearing a 7-(diethylamino)-9,9-dimethylfluoren-2-yl donor group the difference between the excited and the ground state dipole moments, estimated from the Lippert-Mataga expression, reached 20 D, which is among the largest reported for neutral dipolar fluorophores. Moreover, the new dyes are characterized by significant two-photon absorption cross-section (up to 450 GM) and large fluorescence quantum yields. The strong decrease in the fluorescence quantum yields of the dyes in polar protic solvents was observed together with the increase in the non-radiative deactivation rates, which can originate from twisted intramolecular charge transfer and intermolecular proton transfer phenomena. In comparison to the parent 3-hydroxychromone derivatives, the new dyes presented significantly improved photostability, which confirms that photodegradation of 3-hydroxychromones occurs from a product of the excited-state intramolecular proton transfer (phototautomer). Finally, an application of the new dyes for probing local binding site polarity of serum albumin was shown. This new class of fluorescent dyes may serve as attractive building blocks for future molecular sensors utilizing environment-sensitive fluorophores.

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers.

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films.

Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer

Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (−) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(−)PBS annealing mechanism by activating a loop–loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (−)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(−)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can ‘freeze’ PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.