Rachel Auzély-Velty

Designing Hyaluronic Acid-Based Layer-by-Layer Capsules as a Carrier for Intracellular Drug Delivery

Polyelectrolyte microcapsules were prepared by the layer-by-layer assembly of hyaluronic acid (HA) and a polycationic polymer, poly(allylamine) (PAH) or poly(lysine) (PLL). The influence of the polycationic partner on the morphology, stability, permeability properties, and enzymatic degradation of microcapsules was thoroughly analyzed. It was found that these properties could be tuned by shell cross-linking. Confocal microscopy studies of cellular uptake of the capsules showed that the polyelectrolyte shells remain stable outside the cells but readily break open once internalized by cells, suggesting their potential as carrier for intracellular drug delivery.

Design of Biomimetic Cell-Interactive Substrates Using Hyaluronic Acid Hydrogels with Tunable Mechanical Properties

Hyaluronic acid (HA) is a natural polysaccharide abundant in biological tissues with excellent potential for constructing synthetic extracellular matrix analogues. In this work, we established a simple and dependable approach to prepare hyaluronic acid-based hydrogels with controlled stiffness and cell recognition properties for use as cell-interactive substrates. This approach relied on a new procedure for the synthesis of methacrylate-modified HA macromers (HA-MA) and, on photorheometry allowing real time monitoring of gelation during photopolymerization. We showed in this way the ability to obtain gels that encompass the range of physiologically relevant elastic moduli while still maintaining the recognition properties of HA by specific cell surface receptors. These hydrogels were prepared from HA macromers having a degree of methacrylation <0.5, which allows to minimize compromising effects on the binding affinity of HA to its cell receptors due to high substitution on the one hand, and to achieve nearly 100% conversion of the methacrylate groups on the other. When the HA hydrogels were immobilized on glass substrates, it was observed that the attachment and the spreading of a variety of mammalian cells rely on CD44 and its coreceptor RHAMM. The attachment and spreading were also shown to be modulated by the elastic properties of the HA matrix. All together, these results highlight the biological potential of these HA hydrogel systems and the needs of controlling their chemical and physical properties for applications in cell culture and tissue engineering.

Internal Composition versus the Mechanical Properties of Polyelectrolyte Multilayer Films: The Influence of Chemical Cross-Linking

Different types of polyelectrolyte multilayer films composed of poly(L-lysine)/hyaluronan (PLL/HA), chitosan/hyaluronan (CHI/HA) and poly(allylamine hydrochloride)/poly(L-glutamic acid) (PAH/PGA) have been investigated for their internal composition, including water content, ion pairing, and ability to be covalently cross-linked, as well as for their mechanical properties. Film buildup under physiological conditions was monitored by the quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), which allows unambiguous quantification of the different groups present in the polyelectrolytes. (PAH/PGA) films emerged as the most dense films with the lowest hydration (29%) and the highest COO(-) molar density. In addition, PAH is greatly in excess in these films (3 PAH monomers per PGA monomer). The formation of amide bonds during film cross-linking using the water-soluble carbodiimide EDC was also investigated. All of the films could be cross-linked in a tunable manner, but PAH/PGA exhibited the highest absolute number of amide bonds created, approximately 7 times more than for (PLL/HA) and (CHI/HA) films. The Youngs modulus E of the films measured by AFM nanoindentation was shown to vary over 1 to 2 orders of magnitude for the different systems. Interestingly, a linear relationship between E and the density of the covalent cross-links created was observed for (PLL/HA) and (CHI/HA) films whereas (PGA/PAH) films exhibited biphasic behavior. The mean distance between covalent cross-links was estimated to be approximately 11 nm for (PLL/HA) and (CHI/HA) films and only approximately 6 nm for (PAH/PGA) films for the maximum EDC concentration tested (100 mg/mL).

Polyester nanoparticles presenting mannose residues: toward the development of new vaccine delivery systems combining biodegradability and targeting properties.

We report the synthesis of fully biodegradable polymeric nanoparticles presenting mannose residues at their surface and their interaction with lectins. A simple and versatile method was used to reach the surface functionalization of poly(D,L-lactic acid) (PLA) nanoparticles by mannose moieties: It consists in using an amphiphilic mannosylated poly(ethylene oxide)-b-poly(E-caprolactone) (PEO-b-PCL) diblock copolymer as a bioresorbable surface modifier in a simple nanoprecipitation-evaporation procedure. The size and zeta potential of the nanoparticles were found to depend on the molar copolymer/PLA ratio, demonstrating the influence of the copolymer on the formation of the nanoparticles. The bioavailability of the mannose residues as specific recognition sites on the nanoparticle surface could be demonstrated by a modified enzyme-linked lectin assay (ELLA) using biotin-labeled lectins which interact specifically with alpha-D-mannopyrannoside derivatives. Besides specific interaction by lectin-mannose complex formation, nonspecific adsorption of the proteins on the nanoparticle surface was observed. These results were fully supported by isothermal titration calorimetry experiments which suggested that the balance between specific and nonspecific interactions can be controlled by the amount of glycosylated polymer used for the preparation of the nanoparticles. Such nanoparticles are expected to be specifically recognized by mannose receptors, which are highly expressed in cells of the immune system. The targeting properties of these carrier systems combined with their potential adjuvant effects due to their size in the range of 200-300 nm make them attractive candidates as vaccine delivery systems.

Multilayer assembly of hyaluronic acid/poly(allylamine): control of the buildup for the production of hollow capsules.

The objective of this work was to investigate the formation of hollow microcapsules composed of hyaluronic acid (HA) and poly(allylamine) (PAH) by layer-by-layer adsorption on CaCO 3 microparticles and subsequent core removal by addition of chelating agents for calcium ions. We found that the molecular weight of HA as well as the HA solution concentration used during deposition are crucial parameters influencing the multilayer structure. Whereas the effect of molecular weight of HA was mainly attributed to the porous structure of the template which allows penetration of polyelectrolytes when their size is below the maximum pore size of the template ( approximately 60 nm), that of the concentration of the HA solution was related to the intrinsic properties of the polysaccharide. Indeed, as shown by quartz crystal microbalance with dissipation monitoring as well as electron microscopy techniques, the latter leads to dense structures for concentrations from five to ten times the critical overlap concentration during adsorption. Such conditions were found to be favorable for the formation of hollow shells. Regarding conditions for core dissolution, we demonstrated the possibility to use either ethylenediaminetetraacetic acid (EDTA) or citric acid as chelating agents. However, in some cases, it was necessary to chemically cross-link the shell to maintain its integrity.

Liquid crystals with restricted molecular topologies: supermolecules and supramolecular assemblies

The term liquid crystal is often associated with fascinating compounds that exhibit unusual melting or solubilisation properties, however, it also represents a unique collection of mesophases that exist between the solid state and the amorphous liquid. As such, this unique state of matter can be accessed by a wide variety of materials from low molar mass to polymeric systems. In this Feature Article we describe some recent studies concerning the liquid-crystalline behaviour of ‘in-between’ materials that have discrete molecular structures, and which are oligomeric but not low molar mass or polymeric systems. Thus, these materials could be described as having supermolecular architectures. We examine some of their mesophase properties and their abilities to form supramolecular assemblies. The development of liquid crystals that have large molecular structures or consist of large scale assemblies is one step towards creating novel self-organising systems which are of a similar dimension to certain biological materials, such as proteins.

Superselective Targeting Using Multivalent Polymers

Despite their importance for material and life sciences, multivalent interactions between polymers and surfaces remain poorly understood. Combining recent achievements of synthetic chemistry and surface characterization, we have developed a well-defined and highly specific model system based on host/guest interactions. We use this model to study the binding of hyaluronic acid functionalized with host molecules to tunable surfaces displaying different densities of guest molecules. Remarkably, we find that the surface density of bound polymer increases faster than linearly with the surface density of binding sites. Based on predictions from a simple analytical model, we propose that this superselective behavior arises from a combination of enthalpic and entropic effects upon binding of nanoobjects to surfaces, accentuated by the ability of polymer chains to interpenetrate.

Investigation of the complexation of (+)-catechin by β-cyclodextrin by a combination of NMR, microcalorimetry and molecular modeling techniques

(+)-Catechin is a polyphenolic compound of natural origin that presents anti-oxidant properties of interest for therapeutics or cosmetics uses. Preliminary studies on inclusion into cyclodextrin cavities yielded contradictory results both for the quantitative (affinity constant) and qualitative description of the interaction. By a combination of several experimental and theoretical methods, the present study resolved the previous ambiguities about the interaction between (+)-catechin and beta-cyclodextrin. Thermodynamic data measured by isothermal titration calorimetry demonstrate that the binding is enthalpy driven. Excellent agreement has been obtained for the measurement of the association constant by NMR and microcalorimetry. The several docking modes obtained by systematic docking studies have been compared to intermolecular contacts measured by NMR and the overall geometry of the complex can be proposed.

Effect of RGD-functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(L-lysine) (PLL) and poly(L-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.

Designing multivalent probes for tunable superselective targeting

Significance A basic requirement in biomedical research is the ability to specifically target cells and tissues. Targeting typically relies on the specific binding of a “ligand” on a tailor-made probe to a “receptor” on the desired cell/tissue. Conventional probes efficiently distinguish a biological entity displaying the receptor from others that do not, but exhibit limited selectivity when the entities to be distinguished display a given receptor at different densities. Multivalent probes that bind several receptors simultaneously potentially can sharply discriminate between different receptor densities. We demonstrate how such “superselective” binding can be tuned through probe design to target a desired receptor density, and thus lay the foundation for the rational design of a new generation of analytical, diagnostic, and therapeutic probes. Specific targeting is common in biology and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined density of surface binding sites. Here we show, using a combination of experiments and simulations on multivalent polymers, that such “superselective” binding can be tuned through the design of the multivalent probe, to target a desired density of binding sites. We develop an analytical model that provides simple yet quantitative predictions to tune the polymer’s superselective binding properties by its molecular characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biology. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44–hyaluronan interactions.