Methacrylated Cellulose Nanocrystals as Fillers for the Development of Photo-Cross-Linkable Cytocompatible Biosourced Formulations Targeting 3D Printing.

Cellulose nanocrystals (CNCs) from cotton were functionalized in aqueous medium using methacrylic anhydride (MA) to produce methacrylated cellulose nanocrystals (mCNCs) with a degree of methacrylation (DM) up to 12.6 ± 0.50%. Dispersible as-prepared CNCs and mCNCs were then considered as reinforcing fillers for aqueous 3D-printable formulations based on methacrylated carboxymethylcellulose (mCMC). The rheological properties of such photo-cross-linkable aqueous formulations containing nonmodified CNCs or mCNCs at 0.2 or 0.5 wt% in 2 wt% mCMC were fully investigated. The influence of the presence of nanoparticles on the UV-curing kinetics and dimensions of the photo-cross-linked hydrogels was probed and 13C CP-MAS NMR spectroscopy was used to determine the maximum conversion ratio of methacrylates as well as the optimized time required for UV postcuring. The viscoelasticity of cross-linked hydrogels and swollen hydrogels was also studied. The addition of 0.5 wt% mCNC with a DM of 0.83 ± 0.040% to the formulation yielded faster cross-linking kinetics, better resolution, more robust cross-linked hydrogels, and more stable swollen hydrogels than pure mCMC materials. Additionally, the produced cryogels showed no cytotoxicity toward L929 fibroblasts. This biobased formulation could thus be considered for the 3D printing of hydrogels dedicated to biomedical purposes using vat polymerization techniques, such as stereolithography or digital light processing.

Topological defects in polycrystalline hexosomes from ß-cyclodextrin fatty esters.

Colloidal nanoparticles were prepared by aqueous self-assembly of amphiphilic ß-cyclodextrins (ßCDs) acylated on their secondary face with C14 chains to a total degree of substitution of 7.0, via a thermolysin-catalyzed transesterification process. The small-angle X-ray scattering pattern of the nanoparticles was consistent with a reverse hexagonal organization. Cryo-transmission electron microscopy images revealed particles with spectacular tortuous shapes and consisting of misoriented domains with a regular columnar hexagonal structure, separated by sharp interfaces. Edge dislocations as well as a variety of stepped tilt grain boundaries (GBs) composed of symmetrical and asymmetrical sections, together with one twist GB, were identified from axial views of the columnar organization. The tilt GB structure was analyzed using the concepts of coincidence site lattice and structural units developed to describe the atomic structure of interfaces in various types of polycrystals. The tilt GBs were described using sequences of ßCD-C14 columns that differed by the number of neighboring columns (5, 6 or 7) and exhibiting distinctive contrasts. To our knowledge, this is the first time that these types of topological defects are described at the nanometric scale by direct observation of colloidal polycrystalline hexosomes of self-organized amphiphiles.

Honeycomb Organization of Chitin Nanocrystals (ChNCs) in Nanocomposite Films of UV-Cured Waterborne Acrylated Epoxidized Soybean Oil Emulsified with ChNCs.

Stable biobased waterborne Pickering dispersions of acrylated epoxidized soybean oil (AESO) were developed using chitin nanocrystals (ChNCs) as sole emulsifier without any additives. Thin AESO-ChNC nanocomposite films were produced by UV-curing thin-coated layers of the AESO emulsion after water evaporation. The kinetics of photopolymerization were assessed by monitoring the consumption of the AESO acrylate groups by infrared spectroscopy (Fourier transform infrared (FTIR)). The curing was faster in the presence of ChNCs, with a disappearance of the induction period observed for neat AESO. The coating of AESO droplets with a thin layer of ChNCs was confirmed by scanning electron microscopy (SEM) observation. SEM and transmission electron microscopy (TEM) images revealed the honeycomb organization of ChNCs inside the cured AESO-ChNC films. The mechanical, thermal, and optical properties of the nanocomposite films were studied by dynamic mechanical analysis (DMA), tensile testing, differential scanning calorimetry (DSC), and transmittance measurement, as a function of ChNC content. The inclusion of ChNCs is strongly beneficial to increase the stiffness and strength of the cured films, without compromising its optical transparency. The ability of ChNCs to act as an emulsifier for AESO in replacement of synthetic surfactants and their strong reinforcing effect in UV-cured films offer new opportunities to produce waterborne stable dispersions from AESO for application in biobased coatings and adhesives.

Self-Assembly of Amphiphilic Biotransesterified ß-Cyclodextrins: Supramolecular Structure of Nanoparticles and Surface Properties.

A series of ß-cyclodextrin (ßCD) amphiphilic derivatives with varying degrees of substitution were prepared by acylating ßCDs on their secondary face using thermolysin to catalyze the transesterification. After dissolution in acetone, the ßCD-Cn derivatives (n = 8, 10, 12, 14) were nanoprecipitated in water, where they self-organized into structured particles that were characterized using cryo-transmission electron microscopy (cryo-TEM) images and small-angle X-ray scattering (SAXS) data. Two types of morphologies and ultrastructures were observed depending on the total degree of substitution (TDS) of the parent derivative. The molecules with TDS < 5 formed nanospheres with a multilamellar organization, whereas those with TDS > 5 self-assembled into barrel-like (n = 8, 10, 12) or more tortuous (n = 14) particles with a columnar inverse hexagonal structure. In particular, faceted ßCD-C14 particles (TDS = 7) appeared to be composed of several domains with different orientations that were separated by sharp interfaces. Ultrastructural models were proposed on the basis of cryo-TEM images and the analysis of the contrast distribution in different projections of the lattice. Complementary compression isotherm experiments carried out at the air-water interface also suggested that differences in the molecular conformation of the series of derivatives existed depending on whether TDS was lower or higher than 5.

Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans.

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis.

BACKGROUND: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. METHODS: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. RESULTS: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. CONCLUSIONS: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. GENERAL SIGNIFICANCE: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.

Characterization of hyperbranched glycopolymers produced in vitro using enzymes.

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10(6) to 4.3 × 10(7) g mol(-1), which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.

In vitro synthesis of hyperbranched α-glucans using a biomimetic enzymatic toolbox.

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

Time-Dependent Elastic Tensor of Cellulose Nanocrystal Probed by Hydrostatic Pressure and Uniaxial Stretching.

The elastic properties of crystals are fundamental for structural material. However, in the absence of macroscopic single crystals, the experimental determination of the elastic tensor is challenging because the measurement depends on the transmission of stress inside the material. To avoid arbitrary hypotheses about stress transfer, we combine hydrostatic pressure and uniaxial-stretching experiments to investigate the elastic properties of cellulose Iß. Three orthogonal compressibilities are 50.0, 6.6, and 1.71 TPa-1. Combining Poisson's ratios from a uniaxial stretching experiment directly gives the Young's modulus along the chain direction (E33). However, Poisson's ratio depends on the deformation rate leading to apparent modulus E33 = 113 GPa using a slow cycle (hours) and 161 GPa using a fast cycle (minutes). The lattice deformation along the chain is not time-dependent, so the off-diagonal elements are time-dependent on the scale of minutes to hours.

Deletion of BSG1 in Chlamydomonas reinhardtii leads to abnormal starch granule size and morphology.

Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for "Bimodal Starch Granule". Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed.