Injectable hydrogels based on alginates grafted with LCST side-chains of different chemistry.

A homologous series of thermoassociating copolymers was prepared by grafting onto alginates different amounts of three different temperature responsive polymers: poly(N-isopropylacrylamide), poly(di(ethylene glycol)methacrylate) and poly(ethylene oxide-co-propylene oxide). From a large set of analytical techniques combining rheology, calorimetry, NMR and SAXS, the relevant parameters controlling the sol/gel transition and the gel properties, mainly the degree of entanglement of macromolecules and the fraction of responsive stickers, were highlighted and interpreted objectively by considering the particularities of the phase diagrams of LCST polymers. Complementary analyses were implemented to investigate adhesiveness, injectabilty, gel swelling and molecular release in physiological environment of thermogelling formulations. In particular, it is shown that steady shear experiments allow to predict the injection forces by taking into account the characteristics of the system (syringe and needle), and that the rapid gelation of the formulations when they are heated at 37 °C delays the release of small molecules into the environment. The overall set of data is discussed in the framework of scaling relations in order to draw quantitative guidelines for the design of injectable thermoresponsive hydrogels.

Sol/gel transition of thermoresponsive Hyaluronan: From liquids to elastic and sticky materials.

Thermoassociating copolymers were prepared by grafting temperature responsive poly(N-isopropylacrylamide-stat-N-tert-butylacrylamide) telomers onto hyaluronan. By varying the composition of LCST side chains, from 50 to 100 wt% of NIPAM units, it is shown that the sol/gel transition of entangled solutions can be accurately controlled in the range of 10 to 35 °C with an abrupt transition and reversible properties. Complementary experiments, performed by DSC and NMR, demonstrate the close relationship between thermoassociation of LCST grafts, forming microdomains of low mobility, and macroscopic properties. Moreover, by performing tack experiments during heating we demonstrate that hyaluronan formulations abruptly switch from a weak adhesive viscous behavior to an elastic adhesive profile in the gel regime. As LCST side-chains form concentrated micro-domains of low mobility, physical gels can resist to dissociation above their sol/gel transition for relatively long periods when immersed in excess physiological medium. The thermoassociative behavior of these copolymers, whose properties can be finely tuned in order to form sticky gels at body temperature, clearly demonstrates their potential in biomedical applications such as injectable gels for drug delivery or tissue engineering.

Cold gelation of alginates induced by monovalent cations.

A new reversible gelation pathway is described for alginates in aqueous media. From various samples differing by their mannuronic/guluronic content (M/G), both enthalpic and viscoelastic experiments demonstrate that alginates having a high M content are able to form thermoreversible assemblies in the presence of potassium salts. The aggregation behavior is driven by the low solubility of M-blocks at low temperature and high ionic strength. In semidilute solutions, responsive assemblies induce a strong increase of the viscosity below a critical temperature. A true physical gel is obtained in the entangled regime, although the length scale of specific interactions between M-blocks decreases with increasing density of entanglements. Cold setting takes place at low temperatures, below 0 °C for potassium concentrations lower than 0.2 mol/kg, but the aggregation process can be easily shifted to higher temperatures by increasing the salt concentration. The self-assembling process of alginates in solution of potassium salts is characterized by a sharp gelation exotherm and a broad melting endotherm with a large hysteresis of 20-30 °C between the transition temperatures. The viscoelastic properties of alginate gels in potassium salts closely depend on thermal treatment (rate of cooling, time, and temperature of storage), polymer and salt concentrations, and monomer composition as well. In the case of alginates with a high G content, a similar aggregation behavior is also evidenced at higher salt concentrations, but the extent of the self-assembling process remains too weak to develop a true gelation behavior in solution.

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures.

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.

New block-copolymer thermoassociating matrices for DNA sequencing: effect of molecular structure on rheology and resolution.

A new family of matrices for DNA sequencing by capillary electrophoresis is presented. These matrices combine easy injection with high sieving performances, due to thermal switching between a low and a high viscosity state through a modest increase in temperature (approximately 20 degrees C). They are constructed from a hydrophilic polymer backbone with grafted lower critical solution temperature (LCST) side chains. The comb-like LCST copolymers are characterized in terms of size of the polymer backbone, the size of LCST side chains and the grafting densities. The dependance of rheological behavior and electrophoretic performance of these copolymers is correlated with their microstructure. Without complete optimization, a resolution of order 0.5, corresponding to a very reasonable limit for read length with current base calling softwares, could be achieved for segments around 800 bases differing by 1 base in less than one hour in a commercial ABI 310 apparatus.