Enhancement reactivity of Bombyx mori silk fibroins via genipin-mediated grafting of a tyrosine-rich polypeptide

Bombyx mori silk fibroin has potential applications in the biomedical field owing to its satisfactory biocompatibility. In the present work, a tyrosine-rich polypeptide (P) with the sequence of GKGYGGYGK was grafted onto fibroin molecules using a natural cross-linking agent of genipin (GP), aiming at improving the reactivity of the fibroin-based materials. Incubation of the polypeptide-grafted fibroin membrane was subsequently carried out, with a mushroom tyrosinase (MT) and a model functional macromolecule of ε-poly-l-lysine (ε-PL). The changes in the structure and composition of the silk fibroin before and after polypeptide grafting were examined by means of size exclusion chromatography and amino acid analysis. The results indicated that the polypeptide was covalently bonded to fibroin chains and evidently promoted the succeeding enzymatic coupling of ε-PL with fibroins, resulting in a higher content of ε-PL compared to the control. The obtained antibacterial activities against S. aureus and E. coli for the fibroin membrane treated with GP/P and MT/ε-PL was more satisfactory than others. Cytotoxicity testing reveals that polypeptide grafting did less impact on the biocompatibility of silk fibroins. The present work provides a novel method to improve the reactivity of silk fibroins and it can be utilized for bio-functionalization of silk fibroin.     

铝—京尼平结合同步鞣染工艺及革的性能

在前期对有机酸蒙囿铝鞣液的性质以及铝-京尼平(genipin)结合鞣制皮粉的试验基础上,进行了Al-genipin结合鞣生产牛鞋面革的小试—用柠檬酸蒙囿的硫酸铝预鞣,再用genipin复鞣,同时将革染成蓝黑色。结果表明:当Al2(SO4)3·18H2O的用量为软化皮坯的40%(折合Al2O3为6%),genipin的用量为削匀白湿皮的6%时,结合鞣革的收缩温度(Ts)可达到92℃,DSC分析的onset温度为89℃;其柔韧性、手感、粒面平细度等综合物理机械性能以及加脂后革的耐水洗性能,都优于铝-戊二醛结合鞣、荆树皮栲胶-铝结合鞣的革。     

Control and enhancement of permselectivity of membrane‐based microcapsules for favorable biomolecular transport and immunoisolation

The success of membrane‐based, cell‐encapsulating microcapsules depends on the membrane permselectivity that provides efficient inward transport of nutrients, therapeutic protein egress, and complete exclusion of immunoglobulins. Microcapsules with a calcium crosslinked alginate core and a genipin‐crosslinked chitosan alginate (GCA) were prepared with good control over size, membrane thickness and density. Importantly, in this study, we report a novel approach of using three relevant biomolecules and investigating the effects of the membrane characteristics (thickness and density) and microcapsule size on biomolecular mass transport across the GCA microcapsules using mathematical models based on a balance of the chemical potential. Scaling analysis was used to interrelate the membrane thickness, chitosan–alginate reaction rate constant, and diffusion coefficient. The resistance to diffusion of the three biomolecules increased with membrane density and thickness. Interestingly, swelling in the large microcapsules resulted in an increase in permeability, allowing larger biomolecules (immunoglobulin and carbonic anhydrase) to diffuse more readily. In the case of the smaller biomolecule, vitamin B₁₂, a shorter diffusion path length in smaller microcapsules allowed better ingress. When compared with other microcapsules, the GCA microcapsules possess improved permselectivity for them to allow diffusion of small nutrient molecules and proteins, whereas completely excluding antibodies. Also, these results elucidate the importance of membrane properties and microcapsule size to realize favorable transport of biomolecules. © 2011 American Institute of Chemical Engineers AIChE J, 2011     

Release of fluorescent markers from phase‐separated gelatin‐maltodextrin hydrogels

Genipin‐crosslinked gelatin‐maltodextrin phase‐separated hydrogels consisting of gelatin‐continuous or bicontinuous microstructures were developed to regulate swelling and release behavior of four fluorescent markers of varying molecular weights [(fluorescein (332 Da) and FITC‐dextrans (FD) (4000–250,000 Da)]. Bicontinuous hydrogels showed significantly greater swelling than gelatin‐continuous hydrogels under all conditions (at pH 1.5 and 7.4 and three genipin/gelatin crosslinking ratios) (P < 0.05). With both microstructures, fluorescein showed the largest release rate and total release followed by FD 4000 Da, FD 40,000 Da, and FD 250,000 Da (P < 0.05). Marker molecular weight, pH, and crosslink ratio all affected the rate and amount of release. The mode of transport for the solvent and all markers was Fickian or slightly anomalous, with diffusional exponent (n) values ranging from 0.35 to 0.64. These results demonstrated that with the proper combination of crosslink density, solvent pH, and microstructure, hydrogels with a specified swelling behavior may be developed. This, coupled with a marker of appropriate size, can lead to controllable levels and rates of release. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Selective Isolation of Trypsin Inhibitor and Lectin from Soybean Whey by Chitosan/Tripolyphosphate/Genipin Co-Crosslinked Beads

Selective isolation of Kunitz trypsin inhibitor (KTI) and lectin from soybean whey solutions by different types of chitosan beads was investigated. The chitosan beads were co-crosslinked with tripolyphosphate/genipin in solutions at pH 5, 7 or 9 (CB5, CB7, CB9). The maximum adsorption ratios of chitosan beads to KTI and lectin were observed at pH 4.4 and 5.4, respectively; highly selective separation was also demonstrated at these pHs. The adsorption ratios increased with temperature, rising between 5 and 25 °C. CB9 produced the best adsorption ratio, followed by CB7 then CB5. The critical interaction governing absorption of chitosan beads to KTI and lectin could be hydrogen bonding. At pH 9, KTI and lectin desorbed efficiently from CB7 with desorption ratios of 80.9% and 81.4%, respectively. The desorption was most likely caused predominantly by electrostatic repulsion. KTI and lectin can effectively be selectively isolated from soybean whey using this novel separation technique.     

Stereoselective Reduction of 1‐O‐Isopropyloxygenipin Enhances Its Neuroprotective Activity in Neuronal Cells from Apoptosis Induced by Sodium Nitroprusside

Genipin is a Chinese herbal medicine with both neuroprotective and neuritogenic activity. Because of its unstable nature, efforts have been to develop more stable genipin derivatives with improved biological activities. Among the new compounds reported in the literature, (1R)‐isopropyloxygenipin (IPRG001) is a more stable but less active compound compared with the parent, genipin. Here, two new IPRG001 derivatives generated by stereoselective reduction of the C₆=C₇ double bond were synthesized. The 1R and 1S isomers of (4aS,7S,7aS)‐methyl‐7‐(hydroxymethyl)‐1‐isopropoxy‐1,4a,5,6,7,7a‐hexahydrocyclopenta[c]pyran‐4‐carboxylate ( CHR20 and CHR21 ) were shown to be very stable both in high‐glucose cell culture medium and in mice serum at 37 °C. Evaluation using an MTT assay and Hoechst staining showed that CHR20 and CHR21 promote the survival of rat adrenal pheochromocytoma (PC12) and retinal neuronal (RGC‐5) cells from injury induced by sodium nitroprusside (SNP). The neuroprotective effects of CHR20 and CHR21 were greater than both isomers of IPRG001, the parent compounds. These results indicate that reduction of 1‐O‐isopropyloxygenipin enhances its neuroprotective activity without affecting its stability.     

京尼平交联制备枯草杆菌碱性蛋白酶聚集体

本研究利用新型交联剂京尼平制备了枯草杆菌碱性蛋白酶交联聚集体(BAP-CLEAs)。以酶活回收率为指标,确定了BAP-CLEAs制备的最佳条件为:交联剂质量浓度0.50%,交联温度35℃,交联时间12 h,此时BAP-CLEAs的酶活回收率为55.04%。采用扫描电镜及红外光谱对BAP-CLEAs进行表征,结果证明枯草杆菌碱性蛋白酶在京尼平的作用下成功交联。与游离酶相比,BAP-CLEAs的最适p H值向碱性方向偏移,由9.4变为10.3,在较宽的p H范围和温度范围内保持较高的酶活。另外,在2%浓度的酪蛋白底物中重复使用5次后,BAP-CLEAs还能保持86.42%的酶活性。以上催化特性的结果表明,枯草杆菌碱性蛋白酶在京尼平的作用下可成功交联形成酶聚集体,且该交联酶聚集体具有比游离酶更优越的p H稳定性、温度稳定性和重复使用稳定性,有良好的工业应用前景。     

New crosslinkers for electrospun chitosan fibre mats. I. Chemical analysis

Chitosan (CS), the deacetylated form of chitin, the second most abundant, natural polysaccharide, is attractive for applications in the biomedical field because of its biocompatibility and resorption rates, which are higher than chitin. Crosslinking improves chemical and mechanical stability of CS. Here, we report the successful utilization of a new set of crosslinkers for electrospun CS. Genipin, hexamethylene-1,6-diaminocarboxysulphonate (HDACS) and epichlorohydrin (ECH) have not been previously explored for crosslinking of electrospun CS. In this first part of a two-part publication, we report the morphology, determined by field emission scanning electron microscopy (FESEM), and chemical interactions, determined by Fourier transform infrared microscopy, respectively. FESEM revealed that CS could successfully be electrospun from trifluoroacetic acid with genipin, HDACS and ECH added to the solution. Diameters were 267 ± 199 nm, 644 ± 359 nm and 896 ± 435 nm for CS–genipin, CS–HDACS and CS–ECH, respectively. Short- (15 min) and long-term (72 h) dissolution tests (T₆₀₀) were performed in acidic, neutral and basic pHs (3, 7 and 12). Post-spinning activation by heat and base to enhance crosslinking of CS–HDACS and CS–ECH decreased the fibre diameters and improved the stability. In the second part of this publication, we report the mechanical properties of the fibres.     

京尼平与皮胶原的反应性研究

以从栀子属植物的果实中提取的环烯醚萜类化合物京尼平为代表,研究其与皮胶原的反应性。采用UV、TLC等方法并结合已有的文献资料,探讨了京尼平与皮胶原的反应机理。用DSC法分析了京尼平的鞣制效果,并对最佳鞣制条件(京尼平的用量、鞣制时间、鞣液温度及pH值等)进行了筛选。结果表明:用干皮粉质量5%的京尼平,在pH=7·5左右鞣制24h后,其DSC曲线的Tp可达到85℃,同时皮粉被染成深蓝色。因此我们认为,京尼平可以作为皮革工业的一种潜在的、可再生的、清洁高效的鞣剂和染料。