Catherine Ladaviere

Chemical Preparation and Structural Characterization of a Homogeneous Series of Chitin/Chitosan Oligomers

The preparation of a homogeneous series of chitin/chitosan oligomers (chito-oligomers) with the same distribution of degrees of polymerization (DP) ranging from 2 to 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described. This DA-series was obtained according to a two-step chemical process involving (i) the production of a well-defined mixture of glucosamine (GlcN) oligomers obtained by acid hydrolysis of a fully N-deacetylated chitosan and after selective precipitations of the hydrolysis products, and (ii) the partial N-acetylation of the GlcN units of these oligomers from a hydro-alcoholic solution of acetic anhydride in a controlled manner. The characterization of this series of samples with different DAs by proton nuclear magnetic resonance (1H NMR) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed us to determine their average DA and identify the main oligomer structures constituting each mixture. Furthermore, MALDI-TOF MS was particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA for the main DPs from 3 to 7. The modeling of these distributions by means of a binomial law displayed that the chemical N-acetylation of low DP GlcN oligomers, produced in a homogeneous medium, occurs randomly along the oligosaccharide chains in accordance with a statistical (Bernoullian) arrangement. In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP.

Synthesis and structural characterization of chitosan nanogels.

Colloidal physical gels of pure chitosan were obtained via an ammonia-induced gelation in a reverse phase emulsion. The water weight fraction and the chitosan concentration in the water phase were optimized so as to yield nanogels with controlled particle size and size distribution. The spherical morphology of the nanogels was established by transmission electron microscopy with negative staining. Wide-angle X-ray scattering experiments showed that these gels were partially crystalline. The electrophoretic mobilities of the particles remained positive up to pH 7, above which the particles aggregated due to the charge neutralization. From the investigation on the colloidal stability of these nanogels in various conditions (pH, salt concentration, temperature), an electrosteric stabilization process of the particles was pointed out, related to the conformation of mobile chitosan chains at the gel-liquid interface. Therefore, the structure of the nanogels was deduced as being core-shell type, a gelified core of neutralized chitosan chains surrounded by partially protonated chains.

Effect of the polymer nature on the structural organization of lipid/polymer particle assemblies.

The nano-organized LipoParticle assemblies, consisting of polymer particles coated with lipid layers, are investigated with the aim of evidencing the impact of the particle chemical nature on their physicochemical behavior. To this end, these colloidal systems are elaborated from anionic submicrometer poly(styrene) (P(St)) or poly(lactic acid) (PLA) particles, and lipid mixtures composed of zwitterionic 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP). As revealed by various experimental techniques, such as quasielastic light scattering, zeta potential measurements, transmission electron microscopy, and 1H NMR spectroscopy, the features of both LipoParticle systems are similar when cationic lipid formulations (DPPC/DPTAP mixtures) are used. This result emphasizes the major role of electrostatic interactions as driving forces in the assembly elaboration process. Conversely, the assemblies prepared only with the zwitterionic DPPC lipid are strongly dependent on the particle chemical nature. The structural characteristics of the assemblies based on PLA particles are not controlled and correspond to aggregates, contrary to P(St) particles. To understand this specific phenomenon, and to consequently improve the final organization of these assemblies which are potentially of great interest in biotechnology and biomedicine, numerous investigations are carried out such as the studies of the impact of the ionic strength and the pH of the preparation media, as well as the presence of ethanol (involved in the PLA particle synthesis) or the mean size of the lipid vesicles. From the resulting data and according to the nature of spherical solid support, hydrophobic effects, hydrogen bonds, or dipole-dipole interactions would also appear to influence the LipoParticle elaboration in the case of zwitterionic lipid formulation.

Particle assemblies: Toward new tools for regenerative medicine

Regenerative medicine is a demanding field in terms of design and elaboration of materials able to meet the specifications that this application imposes. The regeneration of tissue is a multiscale issue, from the signaling molecule through cell expansion and finally tissue growth requiring a large variety of cues that should be delivered in place and time. Hence, the materials should be able to accommodate cells with respect to their phenotypes, to allow cell division to the right tissue, to maintain the integrity of the surrounding sane tissue, and eventually use their signaling machinery to serve the development of the appropriate neo-tissue. They should also present the ability to deliver growth factors and regulate tissue development, to be degraded into safe products, in order not to impede tissue development, and finally be easily implanted/injected into the patients. In this context, colloid-based materials represent a very promising family of products because one can take advantage of their high specific area, their capability to carry/deliver bio-active molecules, and their capacity of assembling (eventually in vivo) into materials featuring other mechanical, rheological, physicochemical properties. Other benefits of great interest would be their ease of production even via high through-put processes and their potential manufacturing from safe, biodegradable and biocompatible parent raw material. This review describes the state-of-the-art of processes leading to complex materials from the assembly of colloids meeting, at least partially, the above-described specifications for tissue engineering and regenerative medicine.

Synthesis of water-soluble and water-insoluble amphiphilic derivatives of dextran in organic medium

Hydrophobically modified dextrans were prepared by reacting native polysaccharide with 1,2-epoxydodecane in dimethylsulfoxide. Epoxide oligomerization was shown to occur as a secondary reaction when hydroxide ions were used as base catalysts. By adjusting the amount of epoxide in the feed, dextran derivatives with degrees of substitution (DS) between 0% and 164% were obtained. Polymers with DS above 100% were readily soluble in organic solvents like tetrahydrofuran, dioxane and water-saturated chloroform and dichloromethane. Their solution properties in organic solvent were characterized by capillary viscometry. Water-soluble derivatives were compared to other amphiphilic dextrans obtained using a heterogeneous modification in aqueous medium. The effect of modification conditions on substitution pattern was evidenced.

Adsorption of plasmid DNA onto lipid/polymer particle assemblies

In this study, the adsorption of plasmid DNA onto preformed lipid/polymer particle assemblies was investigated. These assemblies, called LipoParticles, with a great potential in the biomedical field, were previously studied and thoroughly characterized. They consist of submicrometer anionic spherical polymer particles composed of either poly(styrene) or poly(lactic acid) which are coated with lipid layers formed by mixtures of zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) in tunable proportions. The plasmid adsorption onto the LipoParticle surface was clearly established by the inversion of the surface charge of the assemblies, fluorescence microscopy observations, and agarose gel electrophoresis. From a quantitative standpoint, adsorption isotherms were found to be independent on the polymer core nature but rather strongly dependent on the adsorbed lipid shell composition. Furthermore, the data were fitted with the Langmuir model providing interesting information about the plasmid adsorption process such as the affinity constants between species, as well as the saturation levels of adsorbed plasmids onto assemblies. Finally, the compaction state of the adsorbed DNA was examined thanks to displacement experiments of a DNA-intercalated fluorescent dye, and found to be tightly related to the proportion of the cationic lipid in the adsorbed lipid layer. DPTAP-rich lipid formulations indeed displayed an important plasmid compaction that could be linked to a high affinity constant and a high amount adsorbed.

Shear thinning three-dimensional colloidal assemblies of chitosan and poly(lactic acid) nanoparticles.

In this study, new materials capable of reversible self-assembly, based on concentrated negatively charged poly(lactic acid) nanoparticles and chitosan, a natural polycationic polymer, were successfully fabricated. Electrostatic interactions between oppositely charged components along with weaker interactions led to the formation of a 3D network. The resulting macroscopic assemblies were characterized by dynamic mechanical measurements, and the influences of various parameters such as chitosan/poly(lactic acid) weight ratio, duration and temperature of the mixture, and molecular weight or chitosan degree of acetylation were studied. Our results showed that the mechanical properties of assemblies were highly dependent on the nanoparticle solid content and chitosan/nanoparticle ratio. In particular, at an optimum weight ratio the colloidal assemblies exhibited remarkable high elastic moduli (about 300 kPa) for a particle solid content of 18% w/w. Thanks to the weak and reversible nature of the interactions, these materials exhibited shear thinning properties, and could instantly recover their cohesiveness at rest. The mode of interactions between PLA particles and chitosan was shown to be in part due to electrostatic interactions, but the cross-linking of chitosan-covered particles was attributed to hydrogen bonding. These materials could be envisaged as good candidates for injectable scaffolds for tissue engineering, taking advantage of the biocompatibility and bioactivity of both components. However, some issues concerning temperature stability must be resolved before applying these colloidal assemblies to cell growth in physiological conditions.