Poly(n-butyl cyanoacrylate) nanoparticles via miniemulsion polymerization (1): dextran-based surfactants.

This study aims at synthesizing polysaccharide-coated poly(n-butyl cyanoacrylate) nanoparticles by miniemulsion polymerization. Because of the high reactivity of n-butyl cyanoacrylate, drastic conditions are required in order to emulsify the monomer in water while limiting its anionic polymerization. However, nanoparticles were successfully obtained by miniemulsion polymerization of butyl cyanoacrylate-in-water emulsions stabilized by amphiphilic dextran derivatives. Their physico-chemical properties were thoroughly investigated as a function of amphiphilic dextran structure and concentration. The substitution degree of the amphiphilic dextran used as stabilizer had little influence on the final properties of the obtained nanoparticles. Particle size decreased with the concentration of amphiphilic dextran in the aqueous phase whereas the hydrophilic layer thickness and the amount of adsorbed polysaccharide were nearly constant in the entire range of concentrations studied.

Biodegradable nanoparticles made from polylactide-grafted dextran copolymers.

Polysaccharide-covered polyester nanoparticles were prepared using the emulsion/solvent evaporation process. The core of the nanoparticles was made either of PLA or of a blend of polylactide and polylactide-grafted dextran copolymer in various proportions. The surface of the nanoparticles was covered by dextran chains via the use of water-soluble polylactide-grafted dextrans as polymeric stabilizers during the emulsification step. The characteristics of the nanoparticles (size, surface coverage, thickness of superficial layer, colloidal stability) were correlated to the structural parameters (length and number of polylactide grafts) of the copolymers as well as to their surface active properties. The complete biodegradability of the nanoparticles was evaluated by considering the rate of hydrolysis of polylactide grafts in phosphate buffer and the rate of enzymatic degradation of dextran backbone by dextranase.

Poly(n-butyl cyanoacrylate) nanoparticles via miniemulsion polymerization. 2. PEG-based surfactants.

This study aimed at synthesizing PEG-coated poly(n-butyl cyanoacrylate) nanoparticles by miniemulsion polymerization. Despite the high reactivity of butyl cyanoacrylate, nanoparticles were successfully obtained by miniemulsion polymerization of butyl cyanoacrylate-in-water emulsions stabilized by three PEG-based surfactants: Brij78, Brij700, and Tween80. Their physico-chemical properties were thoroughly investigated as a function of surfactant structure and concentration and discussed on the basis of results obtained from interfacial tension measurements (dodecane-water). Results clearly showed the influence of the length of the PEG part of the surfactant on the final properties of the particles.

Emulsifying properties of biodegradable polylactide-grafted dextran copolymers.

Amphiphilic glycopolymers, polylactide-grafted dextran copolymers (Dex-g-PLA), were synthesized with a well-controlled architecture obtained through a three-step procedure: partial silylation of the dextran hydroxyl groups, ring-opening polymerization of D,L-lactide initiated from remaining hydroxyl groups, silylether deprotection under very mild conditions. Depending on their proportion in polylactide (PLA), these copolymers exhibited solubility either in water or in organic solvents. The emulsifying properties of these glycopolymers were studied: depending on their PLA-to-dextran ratio, they were able to stabilize either direct or inverse emulsions. Droplet size was related to the amount of amphiphilic copolymer in the continuous phase. The aging mechanism of both direct and inverse emulsions was shown to be Ostwald ripening in the first weeks following preparation. Finally inverse miniemulsion copolymerization of acrylamide and N, N'-methylenebisacrylamide was performed in the presence of an amphiphilic Dex-g-PLA stabilizer. Polyacrylamide hydrogel nanoparticles were prepared in that way.

Nanoparticles of hydrophobically modified dextrans as potential drug carrier systems.

Nanoparticles combining a hydrophobically modified dextran core and a polysaccharide surface coverage were elaborated. Their suitability for applications like drug delivery was evaluated. The selected polysaccharide, dextran, was chemically modified by the covalent attachment of hydrocarbon groups (aliphatic or aromatic) via the formation of ether links. According to the extent of modification, either water-soluble or water-insoluble dextran derivatives were obtained. The latter exhibited solubility in organic solvents like tetrahydrofuran or dichloromethane saturated with water. Water-soluble dextran derivatives were used as polymeric surfactants for the control of nanoparticles surface characteristics. Nanoparticles were prepared either by o/w emulsion or solvent-diffusion methods. The size and surface properties of dextran nanoparticles were correlated to processing conditions. The stability of colloidal suspensions was examined as a function of ionic strength and related to the particle surface characteristics. The redispersability of freeze-dried suspensions without the addition of cryoprotectant was demonstrated. Finally, the degradability of modified dextrans was compared to that of starting dextran, after enzymatic hydrolysis in the presence of dextranase.

Aqueous solutions of native and hydrophobically modified polysaccharides: temperature effect.

Amphiphilic polysaccharides, obtained by the attachment of various hydrocarbon groups onto dextran, are studied in aqueous solutions. The viscosity of their aqueous solutions is examined as a function of concentration and temperature in the range 25-65 degrees C. Varying polymer concentration, viscosity follows a polynomial development of Huggins equation in which the coefficients can be calculated from the Huggins constant determined in the dilute domain (Matsuoka-Cowman equation). For all polymers, the solution viscosity follows an Arrhenius-like variation with temperature. The activation energy of the aqueous solutions is determined as a function of polymer concentration and structural characteristics (nature and amount of grafted hydrocarbon groups). The variation of activation energy with polymer concentration is shown to be consistent with predictions based on the Matsuoka-Cowman equation combined with the equation of Andrade. This conclusion is extended to other polysaccharides using data from the literature.

The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.

Cartilage engineering consists of re-constructing functional cartilage by seeding chondrocytes in suitable biomaterials in vitro. The characteristics of neocartilage differ upon the type of biomaterial chosen. This study aims at determining the appropriate scaffold material for articular cartilage reconstruction using non articular chondrocytes harvested from rat sternum. For this purpose, the use of polysaccharide hydrogels such as alginate (AA) and hyaluronic acid (HA) was investigated. Several ratios of AA/HA were used as well as three derivatives obtained by chemical modification of HA (HA-C18, HA-C12(2.3), HA-C12(2.5)-TEG0.5). Sternal chondrocytes were successfully cultured in 3D alginate and alginate/HA scaffolds. HA retention in alginate beads was found to be higher in beads seeded with cells than in beads without cells. HA-C18 improved HA retention in beads but inhibited the chondrocyte synthesis process. Cell proliferation and metabolism were enhanced in all biomaterials when beads were mechanically agitated. Preliminary results have shown that the chondrocyte neo-synthesised matrix had acquired articular characteristics after 21 days culture.

Amphiphilic derivatives of dextran: adsorption at air/water and oil/water interfaces.

Ionic amphiphilic dextran derivatives were synthesized by the attachment of sodium sulfopropyl and phenoxy groups on the native polysaccharide. A family of dextran derivatives was thus obtained with varying hydrophobic content and charge density in the polymer chains. The surface-active properties of polymers were studied at the air-water and dodecane-water interfaces using dynamic surface/interfacial tension measurements. The adsorption was shown to begin in a diffusion-limited regime at low polymer concentrations, that is to say, with the diffusion of macromolecules in the bulk solution. In contrast, at long times the interfacial adsorption is limited by interfacial phenomena: adsorption kinetics or transfer into the adsorbed layer. A semiempirical equation developed by Filippov was shown to correctly fit the experimental curves over the whole time range. The presence of ionic groups in the chains strongly lowers the adsorption kinetics. This effect can be interpreted by electrostatic interactions between the free molecules and the already adsorbed ones. The adsorption kinetics at air-water and oil-water interfaces are compared.

Hydrophobically modified alginate hydrogels as protein carriers with specific controlled release properties.

Amphiphilic derivatives of sodium alginate, prepared by chemical covalent binding of long alkyl chains onto the polysaccharide backbone via ester functions, form strong hydrogels in aqueous solutions. The shear-thinning and thixotropic behaviors of these hydrogels have been exploited to prepare particles (millimetric beads or microparticles) by dispersion in sodium chloride solutions. This all-aqueous procedure was used for the encapsulation of model proteins, such as bovine serum albumin (BSA) and human hemoglobin (Hb), or of a vaccine protein (Helicobacter pylori (H. pylori) urease). In all cases, the encapsulation yields were very high (70-100%). No release of model proteins was observed in water within several days, in contrast with protein-loaded calcium alginate particles, which exhibit an important release within only a few hours. The controlled release of proteins can, however, be achieved by inducing the dissociation of the physical hydrophobic network. This dissociation has been obtained either by addition of surfactants, acting as disrupting agents of intermolecular hydrophobic junctions, or of esterases such as lipases, which hydrolyze the ester bond between alkyl chains and the polysaccharide backbone. The level of immunization against H. pylori infection in mice, induced by encapsulated urease administrated by either systemic or mucosal routes, was also assessed.

Expression of adhesion molecules and collagen on rat chondrocyte seeded into alginate and hyaluronate based 3D biosystems. Influence of mechanical stresses.

Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.

Cartilage repair: surgical techniques and tissue engineering using polysaccharide- and collagen-based biomaterials.

Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen- and polysaccharide-based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan-rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three-dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell-seeding with genetically-modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation.

Amphiphilic polysaccharides: useful tools for the preparation of nanoparticles with controlled surface characteristics.

Polymeric surfactants obtained by hydrophobic modification of dextran are used as stabilizers for oil-in-water emulsions. The kinetics of interfacial tension decrease is studied as a function of polymer structural characteristics (degree of hydrophobic substitution) and at various polymer concentrations. Several hydrocarbon oils, either aliphatic (octane, decane, dodecane, and hexadecane) or aromatic (styrene), are tested. Kinetics exhibits the same general trends no matter which oil or polymer is considered. The emulsifying properties of the polymeric surfactants are illustrated by the preparation of oil-in-water emulsions. The droplet size at the preparation is correlated to the amount of oil and to the polymer concentration in the aqueous phase. For low polymer/oil ratios, it is shown that the droplet size is limited by the initial amount of polymer. On the contrary, for high polymer/oil ratios, the droplet size seems to level down, indicating that other parameters become predominant. Emulsion aging occurs by Ostwald ripening, and it is demonstrated that the theoretical equation of Lifshitz, Slyozov, and Wagner (LSW) correctly describes the experimental results. The nature of the oil has important effects on emulsion aging, as described by the LSW equation. The aging of emulsions containing oil mixtures is quantitatively described on the basis of the results with pure oils. The influence of polymer chemical structure can be conveniently correlated to interfacial tension results through the LSW equation. On the contrary, the influence of oil volume fraction seems to be overestimated by the usual correction factor, k(phi). The effect of temperature on emulsion aging is finally examined. Miniemulsions stabilized with dextran derivatives are used for the radical polymerization of styrene. Following this procedure, polysaccharide-covered polystyrene nanoparticles are prepared and characterized (size and surface coverage). The size of the particles is directly correlated to that of the initial droplets for styrene volume fractions around 10%. On the contrary, for initial styrene volume fractions around 20%, particles exhibit a larger size than the initial droplets, indicating that coalescence processes take place during polymerization. The amount of dextran at the surface of the particles is determined and compared to the adsorbed amounts resulting from emulsion preparation.

Physical alginate hydrogels based on hydrophobic or dual hydrophobic/ionic interactions: bead formation, structure, and stability.

Hydrophobically associating alginate (AA) derivatives were prepared by covalent fixation of dodecyl or octadecyl chains onto the polysaccharide backbone (AA-C12/AA-C18). In semidilute solution, intermolecular hydrophobic interactions result in the formation of physical hydrogels, the physicochemical properties of which can be controlled through polymer concentration, hydrophobic chain content, and nonchaotropic salts such as sodium chloride. The mechanical properties of these hydrogels can then be reinforced by the addition of calcium chloride. The combination of both calcium bridges and intermolecular hydrophobic interactions leads to a decrease in the swelling ratio accompanied by an increase of elastic and viscous moduli. Beads made of hydrophobically modified alginate were obtained by dropping an aqueous solution of alginate derivative into a NaCl/CaCl2 solution. As compared to unmodified alginate beads, modified alginate particles proved to be stable in the presence of nongelling cations or calcium-sequestering agents. However, evidence is presented for a more heterogeneous structure than that of plain calcium alginate hydrogels with, in particular, an increase in the effective gel mesh size, as determined by partition and diffusion coefficient measurements.

Production of microspheres based on hydrophobically associating alginate derivatives by dispersion/gelation in aqueous sodium chloride solutions.

A new "all aqueous" procedure for the preparation of stable polysaccharide microparticles was developed. The method consists of dispersing a water solution of an amphiphilic alginate derivative (in the current work, alginate substituted with low amounts of dodecyl chains) first fluidified under mechanical stress, into an NaCl solution. The procedure exploits the ability of amphiphilic associative derivatives to form strong hydrogels in the presence of nonchaotropic salts and their shear-thinning/thixotropic properties. Depending on the experimental conditions, the size of the microparticles can be varied from 10 microm to several hundred micrometers. Their mechanical properties can eventually be reinforced by addition of low concentrations of calcium chloride. The resulting microparticles exhibit a better stability than that of plain Ca(2+)-alginate particles, as they are not disrupted when nongelling cations or calcium-sequestering agents are added to the solution. In addition, the particles can be easily redispersed after being centrifuged or freeze-dried.

Physicochemical evaluation of PLA nanoparticles stabilized by water-soluble MPEO-PLA block copolymers.

Different water-soluble MPEO-PLA diblock copolymers with various alpha-methoxy-omega-hydroxyl polyethylene (MPEO) and poly(lactic acid) (PLA) block lengths have been synthesized. Their surface-active properties were evidenced by surface tension (water/air) measurements. In each case the surface tension leveled down above a critical polymer concentration, which was attributed to the formation of a dense polymer layer at the liquid-air interface. The applicability of copolymers as emulsion stabilizers in the preparation of PLA nanospheres by an o/w emulsion/evaporation technique was then investigated. Four copolymers presenting sufficient water solubility and good surfactive properties were used to prepare PLA nanospheres with MPEO chains firmly anchored at the particle surface. The effect of polymer concentration in emulsion on particle size and surface coverage was examined. Whatever the copolymer characteristics, it was found that the optimal concentration to obtain a large amount of MPEO at the particle surface was similar (around 2 g/l). The effect of the copolymer composition on MPEO layer characteristics and on colloidal stability was also evaluated. The conformation of MPEO blocks at the PLA particle surface is discussed in relation to the layer thickness and the surface area occupied per molecule.

Cartilage repair using new polysaccharidic biomaterials: macroscopic, histological and biochemical approaches in a rat model of cartilage defect.

OBJECTIVE: The present study aims at evaluating, in a rat model of cartilage defect, the potential of various polymers as filling and repair biomaterials. The macroscopic and histological observations are compared to biochemical parameters in order to appreciate the pertinence of the latter as suitable criteria in tissue engineering. METHODS: A hydrogel, consisting of hyaluronic acid (HA), covalently substituted by hydrophobic alkyl chains (HA12, HA18) and an alginate sponge, alone (Asp) or combined with HA (AHAsp) or combined with HA and chondrocytes (HYBsp) were evaluated. Cartilage lesions were drilled in femoral trochlea of rats. The analyses were performed on trochlea as well as on patella and condyles. RESULTS: Repairs achieved with hydrogels had a similar macroscopic appearance than those afforded by AHAsp and HYBsp. Best macroscopic and histological scores were obtained with HA18 and HYBsp in comparison with alginate group (P< 0.01 and P< 0.02 respectively). Biochemical evaluations confirmed the presence of similar amounts of proteoglycans in the repaired zones and in the controls, though with different DeltadiC4S/DeltadiC6S ratios and enhanced HA levels. CONCLUSIONS: Hydrogels or sponges proved to be colonized by cells synthesizing a matrix with a high HA content. The matrix obtained eventually turns hyaline and takes over the scaffold. The addition of HA and/or chondrocytes to Asp significantly improves the macroscopic and histological scores (P< 0.05 and P< 0.02 respectively). However, biochemical parameters are significantly different of those evaluated in native cartilage. The present study shows that only biochemical parameters allow to discriminate between various biomaterials in tissue engineering and are essential informations which should be taken into account in addition to macroscopic and histological observations.

Surface activity and emulsification properties of hydrophobically modified dextrans.

Neutral polymeric surfactants were synthesized by covalent attachment of hydrophobic groups (aromatic rings) onto a polysaccharide backbone (dextran). By changing the conditions of the modification reaction, the number of grafted hydrophobic groups per 100 glucopyranose units (substitution ratio) was varied between 7 and 22. In aqueous solution, these polymers behaved like classical associative polymers as demonstrated by viscometric measurements. The associative behavior was more pronounced when the substitution ratio increased. The surface-active properties of the modified dextrans were evidenced by surface tension (air/water) and interfacial tension (dodecane/water) measurements. In each case the surface or interfacial tension leveled down above a critical polymer concentration, which was attributed to the formation of a dense polymer layer at the liquid-air or liquid-liquid interface. Dodecane-in-water emulsions were prepared using the polymeric surfactants as stabilizers, with oil volume fractions ranging between 5 and 20%. The oil droplet size (measured by dynamic light scattering) was correlated to the amount of polymer in the aqueous phase and to the volume of emulsified oil. The thickness of the adsorbed polymer layer was estimated thanks to zeta potential measurements coupled with size measurements. This thickness increased with the amount of polymer available for adsorption at the interface. The dextran-based surfactants were also applied to emulsion polymerization of styrene and stable polystyrene particles were obtained with a permanent adsorbed dextran layer at their surface. The comparison with the use of an unmodified dextran indicated that the polymeric surfactants were densely packed at the surface of the particles. The colloidal stability of the suspensions of polystyrene particles as well as their protection against protein adsorption (bovine serum albumin, BSA, used as a test protein) were also examined.

Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage.

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.

Development and characterization of CyA-loaded poly(lactic acid)-poly(ethylene glycol)PEG micro- and nanoparticles. Comparison with conventional PLA particulate carriers.

Cyclosporin A (CyA) loaded poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) micro- and nanoparticles have been developed using an emulsion-solvent evaporation method. Physico-chemical properties, peptide loading content and in vitro release profiles of these novel CyA carriers were compared with those corresponding to conventional PLA micro- and nanoparticles. Results obtained confirm the previously described disposition of PEG chains on the surface of the PLA-PEG formulations. In addition, they revealed the presence of CyA molecules on the surface of both PLA and PLA-PEG systems. Further determination of the surface chemical composition by electron spectroscopy for chemical analysis (ESCA) allowed us to quantify the amount of CyA in the nanospheres' top layers, this amount being higher for nanoparticles than for microparticles, and higher for the PLA systems than for those based on PLA-PEG. In vitro release experiments revealed that PLA-PEG particles provided a more adequate control of CyA release than conventional PLA micro- and nanoparticles. Physico-chemical characterization of the systems during the release studies showed that the developed PLA and PLA-PEG micro- and nanoparticles were not degraded, which suggest a diffusion-mediated release mechanism. Furthermore, we have hypothesized that the hydrophilic outer shell of PEG provides a stationary layer for the diffusion of CyA.

Protein C-loaded monomethoxypoly (ethylene oxide)-poly(lactic acid) nanoparticles.

This paper deals with the preparation and characterization of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO-PLA) nanoparticles containing protein C, a plasma inhibitor which regulates the mechanism of blood coagulation. Protein C was entrapped in MPEO-PLA nanoparticles using the double emulsion method. The influence of MPEO-PLA copolymers on the different parameters was evaluated: characteristics of protein C-loaded nanoparticles, in vitro release of the protein, evolution of the particle size with incubation time and MPEO release. The nanoparticle size does not depend on copolymer characteristics (MPEO and/or PLA block molecular weight). On the other hand, the efficiency of protein C entrapment is affected by the copolymer characteristics. The burst effect during the protein C release is increased with the hydrophilic character of the copolymer. Moreover, protein C adsorption on the particle surface during its release may be related to MPEO release. Only 25% of the released protein C is active, which clearly illustrates that it is altered during its encapsulation. The optimization of the experimental parameters which disturbed entrapped protein C activity, i.e. sonication time and organic solvent was investigated and has led to a preservation of protein C activity. Then, to optimize its entrapment efficiency, a blend PLA/MPEO-PLA (25/75) was used to prepare nanoparticles. This blend limited burst effect of protein C and its adsorption. However, protein C is only partially released which implicates further investigation for a potential therapeutic use.