Modified properties of alternan polymers arising from deletion of SH3-like motifs in Leuconostoc citreum ABK-1 alternansucrase.

Alternansucrase (ALT, EC 2.4.1.140) catalyses the formation of an alternating 〈-1, 3/1, 6-linked glucan, with periodic branch points, from sucrose substrate. Beyond the catalytic domain, this enzyme harbours seven additional C-terminal SH3-like repeats. We herein generated two truncated alternansucrases, possessing deletions of three and seven adjacent SH3 motifs, giving Δ3SHALT and Δ7SHALT. Δ3SHALT and Δ7SHALT exhibited kcat/Km for transglycosylation activity 2.3- and 1.5-fold lower than wild-type ALT (WTALT), while hydrolysis was detected only in the truncated ALTs, oligosaccharide patterns and polymer glycosidic linkage were similar to that of WTALT. The viscosities of ALT polymers increase by ˜100-fold at 15% (w/v), with gel-like states formed at 12.5, 15.0, and 20.0% (w/v) produced by polymer from WTALT, Δ3SHALT, and Δ7SHALT, respectively. The average nanoparticle sizes of Δ3SHALT and Δ7SHALT polymers were 80 nm, compared to 90 nm from WTALT. In conclusion, even relatively subtle differences in the structure of ALT-produced alternan give rise to profound impact on the glucan polymer physicochemical properties.

pH Variation as a Simple and Selective Pathway for Obtaining Nanoparticle or Nanocapsule Polysaccharides.

The fabrication of polysaccharides to be nanoparticles or nanocapsules is quite specific due to various parameters and factors. The present work demonstrates a simple pathway to selectively prepare the ionic polysaccharide flakes to be nanoparticles or nanocapsules. The systematic studies on the model cases of cationic polysaccharide (i.e., chitosan) and anionic polysaccharide (i.e., alginate) confirm that p Ka is the key point to tune the polysaccharides to be nanoparticles or nanocapsules. When the ionic polysaccharides were in an oil/water emulsion system, the pH close to p Ka leads to the densely packed polysaccharide chains under the hydrogen bond networks, and as a result the cross-link occurs all through the chains to be nanoparticles. On the other hand, when pH was adjusted to the lower or higher than p Ka depending on the types of ionic polysaccharide, the polysaccharide chains are under charge-charge repulsive force, resulting in the alignment of polysaccharide chains to be hollow nanospheres, and at that time the cross-link initiates the formation of nanocapsules. The present work, for the first time, clarifies that pH variation is the key to selectively prepare nanoparticles or nanocapsules, and this is important for delivery systems, coatings, sensors, and so forth.

Chitosan-based polymer hybrids for thermo-responsive nanogel delivery of curcumin.

The purpose of this study is to design and develop thermoresponsive nano-sized hydrogel particles from a natural polymer, chitosan, as smart material platforms for curcumin delivery. Chitosan was used as the backbone material to be grafted with poly-(N-isopropylacrylamide) (pNIPAM) using an EDC/NHS coupling reaction. The conjugated products were characterized by 1H NMR and TGA. Chitosan-grafted pNIPAM (CS-g-pN) nanogels were prepared by a sonication method. The loading of curcumin into the CS-g-pN nanogels was achieved using an incubation method. Size, morphology of nanogels, amounts of curcumin loaded to the nanogels and cellular uptake were investigated by DLS, TEM, fluorescent spectroscopy and confocal microscopy techniques, respectively. A CellTiter-Blue® cell viability assay was performed in NIH-3T3 and HeLa cells to assess the safety while MTT assay was carried out in MDA-231, Caco-2, HepG2, and HT-29 cells for determining cytotoxic effects. Results showed that CS-g-pN with 3-60% degree of modification were simply assembled into spherical nanogel particles with submicron sizes, in which curcumin was encapsulated. The thermoresponsive behavior of each CS-g-pN nanogel formulation differed due to the grafted pNIPAM length and density. The CS-g-pN nanogel formulations were non-toxic towards NIH-3T3 and HeLa cells. Each curcumin-loaded CS-g-pN nanogel formulation could be up taken into NIH-3T3 cell lines and showed the dose-dependent cytotoxicity against tested cell lines. Successful development of this curcumin-loaded nanogel will lead to advanced materials that can be functionalized and optimized for targeted therapy and controlled delivery of small molecules and/or biomolecules for biomedical applications.

Thermo-Magnetoresponsive Dual Function Nanoparticles: An Approach for Magnetic Entrapable-Releasable Chitosan.

Magnetic polymeric nanoparticles can be used for selective binding in a magnetic field. However, as the magnetic nanoparticles (MAG) are stabilized with polymers, the separation of the MAG from the polymer chains after use is difficult. This work proposes a combination of a thermoresponsive polymer with MAG allows for the as-desired simple removal of MAG from the polymer chains. For this, chitosan (CS) was conjugated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and antibody (Ab) together with the physisorbed MAG as a thermo-magneto dual functional material. The key synthesis steps are (i) radical polymerization of NIPAM in the presence of mercaptoacetic acid so that the PNIPAM obtained contains terminal carboxylic acid groups (PNIPAM-COOH), (ii) the CS-N-hydroxysuccinamide water-based system that allows conjugation of CS with PNIPAM-COOH in water at room temperature, and (iii) the weak interaction between MAG and the CS chain. As a model application, CS is conjugated with the antirecombinant Leptospirosis Ab (rLipL32) to allow the selective binding and collection of the target antigen under the dual functions. This is the first demonstration of a simple but effective solution for MAG exclusion from the target molecules and will be practical for diverse applications, such as diagnosis, sensors, filtration, etc.