壳聚糖结构和分子量对凝血作用的研究

本工作研究了不同脱乙酰度和不同分子量壳聚糖、壳聚糖醋酸生理盐水溶液及羧甲基壳聚糖生理盐水溶液的凝血作用。在壳聚糖醋酸生理盐水溶液作用下，红细胞发生聚集和形变，而且低脱乙酰度现象明显，分子量在10^5～10^6范围内影响不十分明显；羧甲基壳聚糖不能使红细胞在低相对离心力下发生沉降，仅有部分叠连现象；而壳聚糖则不能使红细胞发生聚集和形变。血液凝血酶时间(TT)、凝血酶原时间(PT)、活化部分促凝血酶原激酶时间(APTT)和纤维蛋白原浓度(FIB)验证了壳聚糖、壳聚糖醋酸生理盐水溶液以及羧甲基壳聚糖均不激活凝血因子，且脱乙酰度、分子量以及取代基对这些指标也没有明显地影响。动态凝血实验发现低脱乙酰度壳聚糖能够更好地使血液凝固。     

不同脱乙酰度对壳聚糖膜与角膜基质细胞相容性的影响

以分子量为30万．脱乙酰度分别为63．3％、73．7％、83％和97％的壳聚糖制备不同的壳聚糖膜，在不同脱乙酰度的壳聚糖膜上培养兔角膜基质细胞．通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖分子脱乙酰度对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖脱乙酰度越高。壳聚糖膜对细胞的损伤越小。越有利于细胞在膜上的生长和贴附，反之．低脱乙酰度的壳聚糖膜与角膜细胞的相容性较差。     

甲壳素/壳聚糖止血机理及应用

甲壳素通过蛋白质介导黏附血小板,形成的甲壳素/血小板复合物加速血纤维蛋白单体的聚合并共同形成凝块;另一方面,甲壳素诱导红细胞聚集,刺激血管收缩,最终形成血栓,封合伤口.壳聚糖的止血机理基本类似于甲壳素,但两者对红细胞的聚集程度和对补体的激活程度存在一定差异.甲壳素独特的三次结晶结构(包括α型和β型)赋予甲壳素优良的止血功能;壳聚糖的止血效果则与它的分子量、脱乙酰度、质子化程度和物理形式等有关.本文综述了甲壳素和壳聚糖的止血机理及应用情况.     

壳聚糖/鞣花酸/红细胞膜脂止血复合海绵的制备与评价

背景：部分文献报道壳聚糖对严重创伤的止血效果有限,因此以壳聚糖为基础止血剂的促凝血活性还有待进一步增强。 目的：制备一种新型的壳聚糖/鞣花酸/红细胞膜脂复合海绵,评价其促凝血活性和细胞毒性。 方法：通过冻干法制备壳聚糖海绵和壳聚糖乙酸盐海绵,然后再通过静电吸附法制备壳聚糖/鞣花酸/红细胞膜脂复合海绵。血浆复钙时间法观察3种海绵的促凝血活性,并检测3种海绵对SD大鼠肝脏的止血效果及对L929细胞的毒性。 结果与结论：壳聚糖/鞣花酸/红细胞膜脂复合海绵组的血浆复钙时间、出血时间、失血量均显著少于壳聚糖海绵组和壳聚糖乙酸盐海绵组(P < 0.01)。细胞实验显示壳聚糖/鞣花酸/红细胞膜脂复合海绵无细胞毒性。说明壳聚糖/鞣花酸/红细胞膜脂复合海绵具有良好的促凝血活性且无细胞毒性。     

真菌发酵制备生物材料壳聚糖

利用犁头霉属真菌发酵生产壳聚糖。发现培养温度、时间以及 p H值等因素对壳聚糖产量及性质产生明显影响。p H值影响壳聚糖的脱乙酰程度 ,p H值控制在 4 .5可获得 90 %以上的脱乙酰度 ;2 8～2 9℃和 p H值 5.0条件下 ,壳聚糖的收率达到 780 mg/ L ;随培养时间延长 ,产品分子量迅速下降。扫描电镜分析显示 ,发酵法生产的壳聚糖成膜后 ,与酸碱处理甲壳素得到的产品在微观结构上存在显著差别     

6-0-2′-羟丙基甲壳质和壳聚糖的制备及其性能的初步研究

研究了甲壳质经氢氧化钠溶液碱化及与环氧丙烷的醚化反应。在温和条件下合成了低取代度(LDS)的6—0—2′—羟丙基甲壳质和壳聚糖。探讨了不同溶剂和时间下反应产物的脱乙酰度(DDAc)、取代度(DS)和特性粘度([η])的变化。发现DS为0.03～0.06,DDAc为30～50％的产物,具有良好的水溶性和成膜性。异丙醇是合成LDS 6—0—2′—羟丙基甲壳质和壳聚糖的良好溶剂。     

壳聚糖对幽门螺杆菌外膜屏障功能的影响

研究壳聚糖 (即脱乙酰壳聚糖 ,Chi tosan)对Hp外膜屏障功能的影响 ,以探讨壳聚糖抗Hp的作用机制 ,以 6种抑菌作用较好的壳聚糖溶液 ,3种 70 %脱乙酰、3种 88.5 %脱乙酰为试验材料。将Hp标准菌株SydneyStrain 1(SS1)、NCTC116 39、NCTC116 37加入上述壳聚糖溶液中 ,细菌浓度为 10 8CFU ml,将每种壳聚糖分别与Hp菌液加入 2 4孔加样板中 ,每孔 2ml,每种溶液做 5孔 ,在 37℃和微需氧条件下 ,12 0r min振荡 ,4 8h后取出孔内液 ,30 0 0r min离心 10min后 ,取上清液分别测葡萄糖含量、天门冬氨酸氨基转移酶 (AST)和γ 谷氨酰转肽酶(GGT)…     

低取代6-O-羧甲基壳聚糖结构及其抗菌和促进皮肤创面愈合的研究

采用波谱等现代仪器分析方法表征6-O-羧甲基壳聚糖结构和基本性能,并以6-O-羧甲基壳聚糖水溶胶膜为基本模型,研究其抗菌性能和临床促进皮肤创面愈合等效果.结果表明:改性壳聚糖为低取代(取代度为9.5%)、高脱乙酰度(脱乙酰度为83.2%)的碳6位取代羧甲基壳聚糖,其热分解起始温度低于180℃.溶胶膜对大肠埃希氏菌、金黄色葡萄球菌以及铜绿假单胞菌作用60min时的杀灭率分别为＞99.99%、98.54%、＞99.99%.溶胶膜使用时一般无需换药,对浅Ⅱ°烧烫伤、深Ⅱ°烧烫伤、手术切口、取皮区、溃疡等多种创面均具有明显的促愈合、止血、抑菌、控制渗出、减少疤痕形成的作用,且未发现全身及局部致敏反应,亦未见创面组织过度增生现象.     

低取代6-0-羧甲基壳聚糖结构及其抗菌和促进皮肤创面愈合的研究

采用波谱等现代仪器分析方法表征6-O-羧甲基壳聚糖结构和基本性能,并以6-O-羧甲基壳聚糖水溶胶膜为基本模型,研究其抗菌性能和临床促进皮肤创面愈合等效果.结果表明改性壳聚糖为低取代(取代度为9.5%)、高脱乙酰度(脱乙酰度为83.2%)的碳6位取代羧甲基壳聚糖,其热分解起始温度低于180℃.溶胶膜对大肠埃希氏菌、金黄色葡萄球菌以及铜绿假单胞菌作用60min时的杀灭率分别为＞99.99%、98.54%、＞99.99%.溶胶膜使用时一般无需换药,对浅Ⅱ°烧烫伤、深Ⅱ°烧烫伤、手术切口、取皮区、溃疡等多种创面均具有明显的促愈合、止血、抑菌、控制渗出、减少疤痕形成的作用,且未发现全身及局部致敏反应,亦未见创面组织过度增生现象.     

不同脱乙酰度壳聚糖支架制备及降解性能评价

制备不同脱乙酰度壳聚糖支架,采用SEM观察其表面形貌,检测孔隙率,吸水溶胀率,体内、外降解率。结果表明不同脱乙酰度支架均具有高孔隙三维结构。随脱乙酰度增加,孔隙率分别为93.46%、90.02%和86.71%;溶胀率分别为820%、803%和772%;第4周体外降解率分别为30.44%、22.88%和17.10%;体内降解率为57.48%、40.23%和29.53%。其降解率与脱乙酰度呈负相关,体内降解速率快于体外。可通过控制壳聚糖的脱乙酰度大小为软骨缺损修复提供匹配良好的降解支架材料。     

Effect of chitosan molecular weight and deacetylation degree on hemostasis

Comparative studies have been carried out among solid-state chitosan soliquoid, chitosan acetic acid physiological saline solution, and carboxymethyl chitosan physiological saline solution to discover the hemostatic effect of molecular weight (M(w)) and deacetylation degree (DA) of chitosan. It was found that solid-state chitosan and chitosan acetic acid physiological saline solution performed different hemostatic mechanisms. When blood mixed with chitosan acetic acid physiological saline solution, the erythrocytes aggregated and were deformed. The DA, especially a low DA, in the chitosan acetic acid physiological saline solution, had a significant effect on the unusual aggregation and deformation of erythrocytes, compared with the effect of M(w) within a range between 10(5) and 10(6). However, this phenomenon could not be observed in solid-state chitosan soliquoid. Solid-state chitosan with a low DA absorbed more platelets and was more hemostatic. Carboxymethyl chitosan physiological saline solution had nothing to do with the aggregation and deformation of erythrocytes but caused local rouleau. The values of thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration (FIB) were measured after the blood was mixed with solid-state chitosan soliquoid, chitosan acetic acid physiological saline solution, and carboxymethyl chitosan physiological saline solution, separately. The results demonstrated that coagulation factors might not be activated by them.     

Rheological characteristics of semi-dilute chitosan solutions

The present work is concerned with experimental results on rheological characteristic properties of semi-dilute chitosan solutions in weakly acetic acid. The flow measurements were conducted on solutions of chitosan of various deacetylation degrees DD (62.8%–86.7%) with changing experimental conditions such as concentration (up to 5 g/100 mL), temperature (20°C–41°C) and shear rate applied (up to 10³s^?1). Chitosan solutions behave generally as a typical non-Newtonian shear-thinning fluid with a characteristic n-parameter lower than 1. The rheological n-parameter decreases with decreasing deacetylation degree and increasing solution concentration but it increases with increasing temperature. Because of strong intermolecular hydrogen bonding even at low concentration the chitosan macromolecules have a tendency to entangle and to network formation. The density of the molecular entanglements in the chitosan solution depends on concentration, temperature and shear rate applied. An effect of the deacetylation degree on the Huggins viscosity constant related to polymer solvent interaction is found. The most important results refer to the activation energy of viscous flow found from Arrhenius plots. The dependence of the activation energy on solution concentration, shear rate and DD is discussed.     

The effect of the degree of chitosan deacetylation on the efficiency of gene transfection

Chitosans with various degrees of deacetylation were synthesized by acetylation with acetic anhydride. These chitosans were evaluated for efficacy of nanoparticle formation, DNA binding efficiency, morphology, and in vitro and in vivo gene transfection efficiency. DNA binding efficacy was reduced as degree of deacetylation was decreased, therefore requiring an increased +/-ratio to effect complete DNA complexation. For chitosan with a molecular weight of 390 kDa, the +/-ratio to achieve complete DNA complexation for degrees of deacetylation of 90%, 70% and 62% was 3.3:1, 5.0:1, and 9.0:1, respectively. The size and morphology of these nanoparticle formulations were not significantly different. The decreased degree of deacetylation results in a decrease in overall luciferase expression levels in HEK293, HeLa, and SW756 cells due to particle destabilization in the presence of serum proteins. However, intramuscular luciferase expression levels increased with decreasing deacetylation over the time points tested. Degree of chitosan deacetylation is an important factor in chitosan-DNA nanoparticle formulation as it affects DNA binding, release and gene transfection efficiency in vitro and in vivo.     

In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation

A commercially available almond emulsin beta-glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This beta-glucosidase preparation was used to investigate hydrolytic activity on five chitosan samples with different molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this beta-glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate buffer were prepared using the different viscosity grades of chitosan. The specific viscosity, measured after addition of beta-glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie-Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis-Menten kinetics. Apparent Michaelis-Menten parameters and initial degradation rates were calculated and compared to determine the influences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower affinity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples confirms the existence of a chitinase in the almond emulsin beta-glucosidase preparation, and further studies are warranted.     

Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines

Biodegradable polymer scaffolds were prepared from chitosan with varying degree of deacetylation for in vitro culture of human breast cancer MCF-7 cell lines. These polymers were characterized in terms of functional groups by FTIR and swelling properties. Polymers having high degree of deacetylation showed better swelling properties irrespective of the molecular weight. These polymers were biocompatible and non-toxic towards human epithelial MCF-7 cell lines. Attachment kinetics of MCF-7 cell lines on to polymer scaffold was investigated and it was observed that polymer having high degree of deacetylation favored better cell attachment. In CPIII polymer scaffold having 80% degree of deacetylation, a maximum of 1 millions cells per mg pf polymer were adsorbed within 1h. It appears that high swelling and high degree of deacetylation of chitosan helped in better adsorption of cancer cell lines. The cellular morphology of the attached cells on chitosan matrix was similar to that observed with regular plastic culture with the difference that, cells grew as three-dimensional clumps on chitosan matrix. Polymer having high degree of deacetylation not only favored better adsorption but also showed improved cell growth kinetics. Maximum cell concentration of 6.5 x 10(5) cells/ml was achieved in 5 days culture on CPIII polymer scaffold. The glucose consumption and lactate production pattern of the MCF-7 cell lines on chitosan polymer matrix were similar to that observed on cell growth on tissue culture flask. These results indicate that chitosan scaffold having high degree of deacetylation can be used for three-dimensional growth of MCF-7 cancer cell lines. Such in vitro 3D culture of cancer cells can thus be used as a model for the cytotoxic evaluation of anticancer drugs.     

Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristics

The effects of the molecular weight (MW) and degree of deacetylation (DD) of chitosan on chitosan hydrogel beads were characterized, and the entrapment efficiency, release of entrapped lipase, and activity of immobilized Candida rugosa lipase were investigated. Fresh and freeze-dried beads were characterized. A solution of lipase was prepared in a 1.5% (w/v) chitosan and 1% (v/v) acetic acid medium, and then dropped into a tripolyphosphate solution to prepare the beads. The release studies were performed over 36 h. The enzyme activity was assayed using the Sigma lipase activity method. Chitosan with high MW and DD resulted in a higher loading. A lower activity was observed for beads produced with high DD chitosan. MW did not have a marked effect on the activity. The release study revealed that enzyme release increased to a maximum when the bead was manufactured with a low MW and a moderate to high DD chitosan sample. Freeze drying did not affect the release or the activity of the lipase. Chitosan with a high MW and DD can thus improve loading and reduce the release of lipase in these beads. The choice of chitosan can affect the activity normalized for lipase loading, and beads with desirable qualities can be produced.     

Electrospinning of chitosan dissolved in concentrated acetic acid solution

Chitosan nanofibers were electrospun from aqueous chitosan solution using concentrated acetic acid solution as a solvent. A uniform nanofibrous mat of average fiber diameter of 130 nm was obtained from the following optimum condition: 7% chitosan solution in aqueous 90% acetic acid solution was successfully electrospun in the electric field of 4 kV/cm. The aqueous acetic acid concentration higher than 30% was prerequisite for chitosan nanofiber formation, because more concentrated acetic acid in water progressively decreased surface tension of the chitosan solution and concomitantly increased charge density of jet without significant effect on solution viscosity. However, acetic acid solution more than 90% did not dissolve enough chitosan to make spinnable viscous concentration. Only chitosan of a molecular weight of 106,000 g/mol produced bead-free chitosan nanofibers, while low- or high-molecular-weight chitosans of 30,000 and 398,000 g/mol did not. Average fiber diameters and size distribution decreased with increasing electric field and more bead defects appeared at 5 kV/cm or more.     

蝇蛆壳提取壳聚糖的综合评价

目的 壳聚糖具有良好的水溶性、生物相容性、生物降解性、成膜性及止血、愈创、消炎等作用,在医用生物材料应用方面具有广阔前景.蝇蛆的皮中含有大量壳聚糖成分,是获得较纯壳聚糖的方式之一.对蝇蛆提取的壳聚糖创伤敷料进行综合评价.方法 按照国家统一标准对蝇蛆壳聚糖进行理化分析(相对分子质量、脱乙酰度和重金属含量)、抑菌、皮肤致敏、细胞毒性和溶血实验.结果 蝇蛆壳聚糖的相对分子质量为266 ku,脱乙酰度为75％,重金属含量小于10-5,细胞毒性总评级为1级,具有较轻细胞毒性.致敏和溶血实验结果表明,蝇蛆壳聚糖材料不具有致敏反应,材料的溶血率为0.769％,小于5％,即没有溶血现象.此外,蝇蛆壳聚糖还具有抑菌作用.结论 蝇蛆壳聚糖具有较好的综合特性,可以满足生物医药方面的要求.     

壳聚糖相对分子量对壳聚糖膜与角膜基质细胞相容性的影响

以脱乙酰度为95％，相对分子量分别为130KDa、220KDa、300KDa和500KDa的壳聚糖制备不同的壳聚糖膜。以各种壳聚糖膜作为基质，体外培养兔角膜基质细胞，通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖相对分子量对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖相对分子量对壳聚糖膜与角膜基质细胞的相容性具有重要的影响，相对分子量过高或过低的情况下，壳聚糖膜与角膜基质细胞相容性较差，对细胞损伤程度较大，细胞在膜上的贴附、生长能力较差；以相对分子量在200KDa～300KDa之间的壳聚糖制备出的膜与角膜细胞具有较好的相容性，细胞可在膜上长成密集单层，适合作为角膜培养的支架材料。