Autoassemblies of α-Cyclodextrin and Grafted Polysaccharides: Crystal Structure and Specific Properties of the Platelets.

Cyclodextrins (CDs) are a family of oligosaccharides with a toroid shape, which exhibit a remarkable ability to include guest molecules in their internal cavity, providing a hydrophobic environment for poorly soluble molecules. Recently, new types of inclusions of α CDs with alkyl grafted polysaccharide chains (pullulan, chitosan, dextran, amylopectin, chondroitin sulfate...) have been prepared which are autoassembled into micro- and nanoplatelets. We report in this paper an extensive investigation of platelets with different compositions, including their reversible hydration (thermogravimetric analysis), crystalline structure (powder X-ray diffraction), dimensions and shapes (scanning electron microscopy-field emission gun), thermal properties, solubility, and melting (micro-differential scanning calorimetry). The crystalline platelets exhibit layered structures intercalating the polysaccharide backbones and CD complexes hosting the grafted alkyl chains. The monoclinic symmetry of columnar-type crystals suggests a head-to-tail arrangement of the CDs. The platelets have a preferentially hexagonal shape with sharp edges, variable sizes, and thicknesses and sometimes show incomplete layers (terraces). The crystal parameters change upon dehydration. Melting temperatures of platelets in aqueous solutions exceed 100 °C. Finally, we discuss the potential relation between the platelet structure and applications for mucoadhesive devices.

Pickering emulsions with α-cyclodextrin inclusions: Structure and thermal stability.

This paper explores structural, interfacial and thermal properties of two types of Pickering emulsions containing α-cyclodextrin inclusion complexes: on one hand, emulsions were obtained between aqueous solutions of α-cyclodextrin and different oils (fatty acids, olive oil, silicone oil) and on the other hand, emulsions were obtained between these oils, water and micro or nano-platelet suspensions with inclusion complexes of hydrophobically-modified polysaccharides. The emulsions exhibit versatile properties according to the molecular architecture of the oils. Experiments were performed by microcalorimetry, X-ray diffraction and confocal microscopy. The aptitude of oil molecules to be threaded in α-cyclodextrin cavity is a determining parameter in emulsification and thermal stability. The heat flow traces and images showed dissolution, cooperative melting and de-threading of inclusion complexes which take place progressively, ending at high temperatures, close or above 100°C. Another important feature observed in the emulsions with micro-platelets is the partial substitution of the guest molecules occurring at room temperature at the oil/water interfaces without dissolution, possibly by a diffusion mechanism of the oil. Accordingly, the dissolution and the cooperative melting temperatures of the inclusion crystals changed, showing marked differences upon the type of guest molecules. The enthalpies of dissolution of crystals were measured and compared with soluble inclusions.

Water desorption of cassava starch granules: A study based on thermogravimetric analysis of aqueous suspensions and humid powders.

This work reports on water desorption from cassava starch in relation with the structure and conditioning of granules in suspensions or after equilibration in desiccators. The experimental work is performed by thermogravimetric analysis with isothermal and non-isothermal protocols and interpreted to derive the activation energies and desorption frequencies according to the humidity range with no adjustable parameter. The analysis points out the different types of water interacting with the starch granules and relates the drying coefficients to their microscopic structure. The work helps clarifying contradictory and partial results from the literature.

Hexagonal-shaped chondroitin sulfate self-assemblies have exalted anti-HSV-2 activity.

The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its binding to certain glycosaminoglycans naturally present on host cell membranes. We took advantage of this interaction to design biomimetic supramolecular hexagonal-shaped nanoassemblies composed of chondroitin sulfate having exalted anti-HSV-2 activity in comparison with native chondroitin sulfate. Nanoassemblies were formed by mixing hydrophobically-modified chondroitin sulfate with α-cyclodextrin in water. Optimization of alkyl chain length grafted on chondroitin sulfate and the ratio between hydrophobically-modified chondroitin sulfate and α-cyclodextrin showed that more cohesive and well-structured nanoassemblies were obtained using higher α-cyclodextrin concentration and longer alkyl chain lengths. A structure-activity relationship was found between anti-HSV-2 activity and the amphiphilic nature of hydrophobically-modified chondroitin sulfate. Also, antiviral activity of hexagonal nanoassemblies against HSV-2 was further improved in comparison with hydrophobically-modified chondroitin sulfate. This work suggests a new biomimetic formulation approach that can be extended to other heparan-sulfate-dependent viruses.

Gelation and micellization behaviors of pluronic(®) F127 hydrogel containing poly(isobutylcyanoacrylate) nanoparticles specifically designed for mucosal application.

The aim of this investigation is to combine the advantages of pluronic(®) F127 hydrogels and nanoparticles composed of poly(isobutylcyanoacrylate) (PIBCA) core coated with a mixture of chitosan and thiolated chitosan to design novel multifunctional formulation for mucosal application. Nanoparticles offer the advantage of being mucoadhesive while pluronic(®) F127 hydrogel allowed prolonged contact time onto mucosal surfaces. This work highlights an unprecedented comprehensive study on the effect of nanoparticles on gelation and micellization behaviors of pluronic(®) F127 using rheology and micro-calorimetry experiments. Results showed that presence of nanoparticles induced (i) smaller crystal peak of F127, (ii) a decrease of the enthalpy of F127 micellization and (iii) a non-reversibility of micelle formation (during heating ramp) and micelle melting (during cooling ramp). Together, these findings suggest that a part of F127 was not able to associate into micelles and the formation of mixed micelles containing F127 unimers and PIBCA/(chitosan/thiolated chitosan) copolymer and/or PIBCA homopolymer was suspected. The interaction of F127 unimers with nanoparticles resulted from their physical de-structuration as revealed by nanoparticle size measurement. In addition, we found that short polymerization duration of one hour induced more pronounced nanoparticle de-structuration. Twenty-four hour-polymerization of isobutylcyanoacrylate in the presence of chitosan and thiolated chitosan led to more stable nanoparticles when mixed with pluronic(®) F127.

Dehydration, dissolution, and melting of cyclodextrin crystals.

Cyclodextrins are a family of oligosaccharides with a toroid shape that exhibit a unique ability of entrapping guest molecules in their internal cavity. Water is the primary guest molecule and is omnipresent in the crystalline phases stabilizing the overall architecture. Despite the presence of water molecules inside the cavity, cyclodextrins provide a hydrophobic environment where poorly soluble molecules can easily fit. In this investigation we put in evidence different types of water in the hydrated α-, ß-, and γ-cyclodextrin crystals. Thermogravimetric measurements identify various binding sites of water and highlight the difference between the crystals equilibrated under various humid atmospheres. We establish by microcalorimetry the limit of solubility versus temperature and measure for the first time the melting temperatures of the hydrated crystals. Dissolution and melting enthalpies are derived and the solubility curves are compared to existing literature. The specific features of each cyclodextrin are underlined.

Clotrimazole-loaded nanostructured lipid carrier hydrogels: thermal analysis and in vitro studies.

The aim of the present work is to design a new formulation containing clotrimazole (CTZ) loaded into nanostructured lipid carriers (NLC) for the treatment of fungal vaginal infections. In order to obtain formulations with suitable viscosity for mucosal application, NLC containing CTZ produced by the ultrasonication method were viscosized by the addition of poloxamer P407 in the NLC dispersion (CTZ-NLC-gel). These systems exhibit well-known thermogelling properties. The rheological characterization of the CTZ-NLC hydrogel using a controlled stress rheometer evidenced that the presence of NLC or CTZ did not affect gelling temperature (Tgel). Dilution with simulated vaginal fluid (SVF) increased the Tgel from 17.4 to 29.6°C. For these thermogelling systems, micro-calorimetric assays conducted by a Micro-DSC III confirmed that the hydrogel-containing CTZ-NLC was able to change its structure with a rapid passage from non-crystalline (liquid) to crystalline (semi-solid) form. Furthermore, when a local application is considered, no drug should pass through the vaginal mucosa, limiting thus the systemic diffusion and toxicity. For this purpose, Franz cell has been employed to investigate the ex vivo permeation of CTZ through pig vaginal mucosa. The results showed no CTZ diffusion. The toxicological experiments performed on HeLa cells after a 24h incubation time confirmed that CTZ-NLC-gel at a concentration of 1mg/mL showed a low toxicity profile resulting in a cell vitality of 77.2%. Interestingly, anti-candida activity studies demonstrated that CTZ-NLC gel was 4-fold more active than Fungizone(®) against Candida albicans. These encouraging results suggest that the hydrogel containing CTZ-NLC could be proposed as an innovative system to administer CTZ to treat vaginal infections.

Note on the formulation of thermosensitive and mucoadhesive vaginal hydrogels containing the miniCD4 M48U1 as anti-HIV-1 microbicide.

The miniCD4 M48U1 was formulated into thermosensitive and mucoadhesive pluronic(®) hydrogels as anti-HIV-1 microbicide. The release kinetics of M48U1 from F127/HPMC (20/1 wt%) and F127/F68/HPMC (22.5/2.5/1 wt%) studied during 24h by using Franz diffusion cells showed that HEC hydrogel (1.5 wt%) used as control released 93% of the peptide, while about 25% of M48U1 remained in pluronic(®) hydrogels. The formulation of M48U1 in pluronic(®) hydrogels ensures a local delivery because no diffusion of the peptide was detected through vaginal Cynomolgus macaque mucosa using Ussing chamber. Finally, toxicological studies showed no significant difference in the HeLa cell viability of the pluronic(®) hydrogels in comparison with HEC and phosphate buffer saline.

Nanostructured fluids from pluronic® mixtures.

Micellization and gelation of binary mixtures of EO99PO69EO99 (pluronic(®) F127) and EO80PO27EO80 (pluronic(®) F68) in aqueous solutions were investigated by means of micro-differential scanning calorimetry and rheology and for a total copolymer concentration fixed at 20 wt%. The aim of this investigation is to determine the interplay between micellization and macroscopic gelation of the mixed solutions. Micro-DSC reveals the formation of two distinct populations in F127/F68 mixtures during heating and subsequent cooling of the solutions. The enthalpies of micellization of each copolymer and the respective onset temperatures remained constant after mixing indicating the predominance of two independent processes of micellization in the mixtures. The F127 exhibits a crystallization transition, at a distinct temperature which persists, but increases in the mixtures with concentrations higher than 10 wt%. Rheological measurements were performed during heating ramps or after maturation periods versus frequency. They showed two types of gelation transitions: either a steep increase of the storage and the loss moduli, which corresponds to the crystallization temperature of the F127 micelles or a progressive jamming transition when no crystal can form. Maturation process has a major effect on the rheological properties of the mixed gels, possibly related to local rearrangements of the two micellar phases. This investigation highlights the unique features of the binary pluronic(®) mixtures, compared to dilution effects of single component aqueous solutions.

The counterbalanced effect of size and surface properties of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles on mucoadhesion due to pluronic F68 addition.

PURPOSE: To evaluate of the effect of size and surface characteristics of poly(isobutylcyanoacrylate) nanoparticles coated with pluronic F68 and thiolated chitosan on mucoadhesion. METHODS: Nanoparticles were obtained by radical emulsion polymerization in presence of different amounts of F68 (0-4%w/v). Mucoadhesion was ex vivo evaluated by applying nanoparticle suspension on rat intestinal mucosa and quantifying the amount of attached nanoparticles after incubation. RESULTS: F68 unimers added in the polymerization medium allowed decreasing nanoparticle size from 251 to 83 nm, but resulted in nanoparticle surface modification. The amount of thiolated chitosan onto nanoparticle surface was decreased resulting in lower thiol groups and zeta potential. Consequently, the decrease of nanoparticle hydrodynamic diameter resulted in eight-fold-increase of the number of nanoparticles attached to the mucosa but a significant decrease of the weight of attached nanoparticles was observed. This unexpected result was due to a decrease of the amount of chitosan and thiolated chitosan available to interact with mucus upon addition of F68 in the polymerization medium. CONCLUSIONS: Addition of F68 should not be recommended to improve the amount of mucoadherent nanoparticles. Further studies could allow understanding if the low amount of small size nanoparticles could be able to improve oral bioavailability.

Formulation of mucoadhesive vaginal hydrogels insensitive to dilution with vaginal fluids.

The main objective of this work was to design thermosensitive and mucoadhesive vaginal hydrogels able to keep their rheological and mucoadhesive properties after dilution with vaginal fluids. Formulations were composed of pluronic F127 or a mix of two pluronics F127 and F68. Both formulations contained hydroxypropylmethyl cellulose (HPMC) as a mucoadhesive polymer. The determination of gelling temperature (T(gel)) after dilution with simulated vaginal fluid (SVF) demonstrated that hydrogels were resistant to dilution and T(gel) values were close to 30°C. Ex vivo mucoadhesion experiments conducted on porcine vaginal mucosa founded on the technique of traction of the adhesive/adherent joint allowed the characterization of mucoadhesive properties of hydrogels by measuring work of adhesion (W) and maximum force of detachment (F(max)). In the case of F127-based hydrogels, W and F(max) were lowered after dilution with SVF. However, in the case of F127/F68-based hydrogels, W, F(max) and mucoadhesion profiles were weakly affected by dilution. These differences could be attributed to the higher elasticity of F127/F68/HPMC (22.5/2.5/1% w/w) hydrogel in comparison with F127/HPMC one (20/1% w/w). Indeed, rheological analyses of the formulations showed that both elastic (G') and viscous moduli (G'') were higher for F127/F68/HPMC (22.5/2.5/1% w/w) than for F127/HPMC hydrogel (20/1% w/w). However, we demonstrated that the higher elasticity of the hydrogel was due to the higher total pluronic concentration and not due to the presence of F68 in the formulation.

What can isothermal titration microcalorimetry experiments tell us about the self-organization of surfactants into micelles?

The aim of the present review is to give a concise analysis of the thermodynamic parameters obtained from isothermal titration microcalorimetry (ITC) experiments for the characterization of the self-organization of surfactants into micelles. This review is also focused on works describing some methods allowing to overcome ITC limitation and to extract accurate thermodynamic values from ITC data.

Mechanisms of micellization and rheology of PEO-PPO-PEO triblock copolymers with various architectures.

Micelle formation was followed by micro-DSC and rheology for aqueous solutions of two copolymers of PEO-PPO-PEO, the Pluronic F127 (from BASF) and the EG56 (from PolymerExpert), a branched copolymer built with three chains of F127 type. It is shown that micellization is endothermic and that, for both polymers, the enthalpy of formation/melting is proportional to total concentration. The rheology of the solutions was carefully analyzed, before gelation for F127, and it reveals firstly the progressive changes of solubility of the unimers (decease of relative solution viscosity), followed by micelle formation over a 10 degrees C range. In this range, the micelle concentration dependence on temperature was deduced from enthalpy measurements and the corresponding volume fractions were derived. Viscosity was interpreted within the framework of well-known theories for hard sphere suspensions (Krieger-Dougherty or Quemada) based on an analogy between micelles and nanosized hairy grain suspensions. The gel state is achieved due to formation of the colloidal crystal. For EG56, the rheology is quite different; as the aggregation increases with temperature, a progression is observed from Newtonian to visco-elastic liquid. The characteristic frequency, defined by the relation G(') = G(''), for EG56 varies with temperature and the corresponding times increase by two orders of magnitude according to an Arrhenius law. The frequency dependence of G(') and G('') at different temperatures can be superposed with a horizontal shift factor and a small amplitude adjustment. There is no elastic solid formation in this case. The "gelation" of these two copolymers is compared to the physical gelation of cold-set gels (gelatin).

Influence of decavanadate clusters on the rheological properties of gelatin.

The influence of polyoxovanadate clusters ([H(2)V(10)O(28)](4-)) on the thermo-reversible gelation of porcine skin gelatin solution (type A, M w approximately 40 000 g.mol (-1), pH = 3.4 << isoelectric point (IEP) approximately 8) has been investigated as a function of temperature and vanadate concentration by combining rheology and microcalorimetry. This work shows that the rheological properties of the system depend on electrostatic interactions between [H(2)V(10)O(28)](4-) and positively charged gelatin chains. In a first stage, we describe the renaturation of the gelatin triple helices in the presence of decavanadate clusters. We reveal that, when gelatin chains are in coil conformation (30 degrees C < T < 50 degrees C), the inorganic clusters act as physical cross-linkers that govern the visco-elastic properties of the mixture with an exponential dependence of the (G', G'') modulus with the vanadate concentration. Below 30 degrees C, we show that gelatin triple helix nucleation is slightly favored by the presence of vanadate, but above a helix concentration of 0.012 g.cm (-3), G' is fully governed by the helix concentration. During the melting process, we reveal the non-fully reversible behavior of the vanadate/gelatin rheological properties and the stabilization of gelatin triple helices due to vanadate species until 50 degrees C. This non-reversible character has also been observed in the same experimental conditions with collagen/vanadate solutions. This is the first time that such a stabilization of triple helices has been reported in the case of gelatin hydrogels chemically cross-linked or not. We propose to analyze these results by considering that triple helix aggregates should persist because of decavanadate bridging, that the nucleation of an extended triple helix network may induce a strong modification of the vanadate cross-linker distribution in the system, or both, thus promoting the formation of thermally stable vanadate/gelatin micro-gels in the dangling end of the triple helices.

First example of biopolymer-polyoxometalate complex coacervation in gelatin- mixtures.

The first example of complex coacervation between a biopolymer and polyoxometalate clusters is identified in the gelatin-decavanadate system.

Gelatin hydrogels cross-linked with bis(vinylsulfonyl)methane (BVSM): 1. The chemical networks.

This paper deals with chemical gelation of gelatin in the presence of a cross-linker, bis(vinylsulfonyl)methane (BVSM), which is able to create covalent C-N bonds with amine groups. The investigation is performed at 40 degrees C, where no triple helices are present. Gelatin is in random coil conformation. The influence of various parameters (gelatin concentration, cross-linker concentration, and pH (number of reacting sites along the gelatin chain)) was examined. Gel formation was followed by rheological and thermodynamic measurements (microcalorimetry) versus time (kinetic measurements). Furthermore, the storage moduli were compared to the number of links formed in the course of gelation. The experiments show that, within the experimental range investigated, a fully homogeneous network is not reached; the chemical gels, even upon completion of the reactions, are still in the critical domain, near the threshold. A power law behavior was put in evidence for the shear modulus versus the distance to the gel point, expressed as the concentration of links per gelatin chain. The exponent (f = 3.4 +/- 0.3) is close to that expected for the vulcanization of long chains. The storage moduli can be superposed on a single curve where the abscissa is the product of the number of C-N links per unit volume and the gelatin concentration at an exponent equal to -0.76 +/- 0.03. This exponent suggests the role of entanglements for interchain cross-linking.

Gelatin hydrogels cross-linked with bisvinyl sulfonemethyl. 2. The physical and chemical networks.

This paper deals with the physical and the chemical gelation of gelatin in the presence of a reactant, bisvinyl sulfonemethyl (BVSM). The strategy of this investigation is to separate the contributions of the two types of cross-links in order to deduce the resultant elasticity of the network. In addition, the question raised by several authors concerning an increase of the thermal stability of the triple helices in the presence of cross-links was examined by using several techniques. In this study, the concentration of gelatin and BVSM were kept constant, while the influence of the thermal protocols was put in evidence. The gel formation was followed by rheological, thermodynamic (microcalorimetry), and optical spectroscopy (optical rotation) measurements. The results demonstrate the large differences which arise on the storage moduli by changing the thermal protocols. Cross-linking of the networks in the presence of the triple helices induce a heterogeneous repartition of the bonds, which can form along the triple helices and at the end of the sequences. Consequently, the rubber like network obtained by denaturation of the triple helices is still reminiscent of the initial twist of the chains, and a large modulus is observed, as if rigid segments were still present (storage modulus 10 times larger than for random cross-linking). The hydrogels have an elastic modulus which is larger that the addition of the physical and chemical contributions. The interpretation of the network elasticity is based on the predominant role of the rigid rods of triple helices, where the BVSM cross-links can either modify the ratio between the apparent length and distance between rods, l/d, and/or increase the rigidity of the interchain connections, which are loose coils for the physical gels. The hydrogels investigated have a network which is still close to the percolation threshold of the physical gel, and therefore, the statistical models known for well developed networks cannot be directly validated in these experimental conditions.

Influence of weak and covalent bonds on formation and hydrolysis of gelatin networks.

The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.