Preparation and characterization of co-processed starch/MCC/chitin hydrophilic polymers onto magnesium silicate

It was aimed to investigate the compressibility, compactibility, powder flow and tablet disintegration of a new excipient comprising magnesium (Mg) silicate co-processed (5%–85% w/w) onto chitin, microcrystalline cellulose (MCC) and starch as the hydrophilic polymers of interest. Initially, the mechanism of tablet disintegration was studied by measuring water infiltration rate, moisture sorption, swelling capacity and hydration ability. Moreover, the powders compression behavior was carried out by applying Kawakita model of compression analysis in addition to porosity and radial tensile strength measurements. In vitro drug release of compacts made of 400?mg ibuprofen and 300?mg of the hydrophilic polymers containing 30% w/w Mg silicate co-precipitate was investigated in phosphate buffer (pH 7.8). This work demonstrated that the incorporation of Mg silicate to the hydrophilic polymers lead to the improvement of powder flowability, compactibility, stability (with regard to storage conditions), compacts crushing strength, and disintegration time in addition to faster drug release. The overall findings are practically advantageous in the context of finding a low cost and multifunctional co-processed excipient of natural origins.     

Glycol chitin/PAA hydrogel composite incorporated bio-functionalized PLGA microspheres intended for sustained release of anticancer drug through intratumoral injection

Abstract

A novel anti-hepatoma drug release hybrid system is prepared by using poly(acrylic acid) (PAA) and glycol chitin as substrate in combination with Paclitaxel (PTX)-loaded bio-biofunctionalized poly(lactic-co-glycolic acid) (PLGA) micro-particles, which is intended for cancer therapy through intratumoral injection. The rheological behavior of glycol chitin (7?wt%)/PAA illustrates that it has low gelling temperature (i.e. 17?°C at pH 7.56) which ensures that the formulation turns to gel at physiological condition. The gelling time of glycol chitin/PAA is 16?minutes at 25?°C and 3?minutes at 37?°C, which is convenient for doctors to inject the in-situ gel formulations into the tumor location of patient. The drug release behavior reveals that the system can dramatically postpone the drug release. The cell viability test indicates that the micro-particles with drug still have 62% inhibitory effect on hepatoma cells in the fourteenth day after combing with hydrogel. This system is a promising approach for cancer therapy through intratumoral injection of in-situ gel formulations to extend retention time at tumor sites.     

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile₁₂₂ in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)₈ induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)₈-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNβ1,4GlcNAc)₄, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.Plants have the ability to detect potential pathogens through the recognition of microbe-associated molecular patterns (MAMPs; also known as pathogen-associated molecular patterns), such as flagellin, elongation factor Tu (EF-Tu), peptidoglycan, LPS, chitin, and β-glucan, which are typical molecular signatures for whole classes of microbes (1, 2). MAMP-triggered defense is the first barrier to prevent the invasion of pathogens and plays a major role in the basal resistance of plants against various pathogens. It is also well known that this defense system is strikingly similar to the innate immunity of animals (1, 3, 4).Leucine-rich repeat receptor-like kinases, flagellin-sensetive 2 (FLS2), and EF-Tu receptor, have been shown to recognize bacterial flagellin and EF-Tu, respectively, and serve as receptors for these MAMPs (5). On the other hand, two types of lysin motif (LysM) proteins, CEBiP (chitin-elicitor binding protein) and CERK1 (chitin-elicitor receptor kinase-1), were identified as the cell-surface receptor for chitin, a representative fungal molecular pattern (6–8). Knockout/-down experiments of these genes showed that both of these LysM proteins are required for chitin perception and signaling in rice, whereas CEBiP-type molecules are not involved in chitin signaling in Arabidopsis, indicating the difference between the chitin receptor systems in these model plants (8). Additionally, another LysM receptor-like kinase, LYK4, was also indicated to contribute to chitin signaling in Arabidopsis (9). In the case of rice, it was also shown that CEBiP and Oryza sativa (Os)CERK1 form a heterooligomeric receptor complex ligand dependently (10).Both CEBiP and OsCERK1 have LysMs, which have been known to bind peptidoglycan and chitin (11), in their ectodomains. In Arabidopsis, CERK1 was shown to bind chitin and trigger immune responses as a kind of “all-in-one” receptor. On the other hand, CEBiP seems to play a major role in the perception of chitin in rice, as the knockdown of CEBiP almost abolished the binding of a radio-labeled chitin oligosaccharide to the plasma membrane, whereas OsCERK1 was shown not to bind chitin (6, 12). Liu et al. recently reported that two other CEBiP homologs, OsLYP4 and -6, also bind chitin and contribute to chitin responses and disease resistance in rice (13), although it is not clear to what extent these proteins contribute as the cell surface receptor for chitin oligosaccharides.It was also shown that the perception of peptidoglycan in Arabidopsis requires CEBiP-like molecules (14). Arabidopsis homologs of CEBiP, LYM1 and LYM3, play a major role for the binding of peptidoglycan and activation of downstream defense responses through the receptor kinase, CERK1. These results showed that the receptor kinase CERK1 is required for both chitin and peptidoglycan signaling, at least in Arabidopsis. The peptidoglycan receptor system in Arabidopsis seems similar to the rice chitin receptor for the requirement of a binding protein and a receptor kinase, although the receptor complex formation by these two proteins was not confirmed.Thus, the detailed analysis of ligand recognition by these CEBiP-like molecules and succeeding formation and activation of receptor complex is critically important to understand the molecular mechanisms leading to the activation of downstream defense responses triggered by these MAMPs. Such information would also contribute to the design of novel receptor molecules suitable for future biotechnological application. We show herein the results obtained by biochemical studies on the binding site of CEBiP, epitope mapping of chitin oligosaccharides by saturation transfer difference (STD) NMR spectroscopy, and molecular modeling/docking studies combined with site-directed mutagenesis of the ectodomain of CEBiP. These results clearly indicated that two CEBiP molecules simultaneously bind to one N-acetylchitoheptaose/octaose, (GlcNAc)_7/8, through a binding site located in the central LysM region of the ectodomain, resulting in the dimerization of CEBiP. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and OsCERK1.     

Spiral and target patterns in bivalve nacre manifest a natural excitable medium from layer growth of a biological liquid crystal

Nacre is an exquisitely structured biocomposite of the calcium carbonate mineral aragonite with small amounts of proteins and the polysaccharide chitin. For many years, it has been the subject of research, not just because of its beauty, but also to discover how nature can produce such a superior product with excellent mechanical properties from such relatively weak raw materials. Four decades ago, Wada [Wada K (1966) Spiral growth of nacre. Nature 211:1427] proposed that the spiral patterns in nacre could be explained by using the theory Frank [Frank F (1949) The influence of dislocations on crystal growth. Discuss Faraday Soc 5:48–54] had put forward of the growth of crystals by means of screw dislocations. Frank''s mechanism of crystal growth has been amply confirmed by experimental observations of screw dislocations in crystals, but it is a growth mechanism for a single crystal, with growth fronts of molecules. However, the growth fronts composed of many tablets of crystalline aragonite visible in micrographs of nacre are not a molecular-scale but a mesoscale phenomenon, so it has not been evident how the Frank mechanism might be of relevance. Here, we demonstrate that nacre growth is organized around a liquid-crystal core of chitin crystallites, a skeleton that the other components of nacre subsequently flesh out in a process of hierarchical self-assembly. We establish that spiral and target patterns can arise in a liquid crystal formed layer by layer through the Burton–Cabrera–Frank [Burton W, Cabrera N, Frank F (1951) The growth of crystals and the equilibrium structure of their surfaces. Philos Trans R Soc London Ser A 243:299–358] dynamics, and furthermore that this layer growth mechanism is an instance of an important class of physical systems termed excitable media. Artificial liquid crystals grown in this way may have many technological applications.     

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated‐chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3‐A1, BmCPAP3‐A2, BmCPAP3‐B, BmCPAP3‐C, BmCPAP3‐D1 and BmCPAP3‐D2) were cloned and expressed in Escherichia coli and purified using metal‐chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3‐D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3‐D1 was similar to BmCPAP3‐A1 and BmCPAP3‐C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin‐binding protein, BmCPAP3‐D1, which exhibits high binding affinity to deacetylated chitin.     

氨基葡萄糖盐酸盐稳定性及其制备的研究

测定了Ｄ－氨基葡萄糖盐酸盐在酸液中的溶解度及在加热条件下其稳定性，探讨了甲壳素酸水解和甲壳素质量与产品质量、收率之间的关系，对工业化生产Ｄ－氨基葡萄糖盐酸盐的工艺条件优化、质量和收率的提高具有指导意义。     

顺铂壳聚糖微球制备工艺的研究

选择壳聚糖为材料，用乳化化学交联技术制备顺铂壳聚糖微球，研究了影响微球制备的因素，在此基础上选择壳聚糖浓度（因素Ａ）、水／油相体积比（因素Ｂ）、搅拌速度（因素Ｃ）、药物与鞯体材料用量比（因素Ｄ）、油相类型（因素Ｅ）、壳聚糖种类（因素Ｆ）及固化时间（因素Ｇ）七个因素，每个因素选择三水平，用正交实验设计安排实验，并以微球 载药量，药物包封率，粒子分布百分数为指标优化微球的制备工艺。