Quantum-dot-assisted fluorescence resonance energy transfer approach for intracellular trafficking of chitosan/DNA complex

Fluorescence resonance energy transfer (FRET) was employed to monitor the molecular dissociation of a chitosan/DNA complex with different molecular weights of chitosan. Chitosan with different molecular weights was complexed with plasmid DNA and the complex formation was monitored using dynamic light scattering and a gel retardation assay. As the chitosan molecular weight increased, a more condensed complex was prepared at various ratios of chitosan to DNA. Plasmid DNA and chitosan were separately labeled with quantum dots and Texas red, respectively, and the dissociation of the complex was subsequently monitored using confocal microscopy and fluorescence spectroscopy. As the chitosan molecular weight in the chitosan/DNA complex increased, the Texas red-labeled chitosan gradually lost FRET-induced fluorescence light when HEK293 cells incubated with chitosan/DNA complex were examined with confocal microscopy. This suggests that the dissociation of the chitosan/DNA complex was more significant in the high molecular weight chitosan/DNA complex. Fluorescence spectroscopy also monitored the molecular dissociation of the chitosan/DNA complex at pH 7.4 and pH 5.0 and confirmed that the dissociation occurred in acidic environments. This finding suggests that the high molecular weight chitosan/DNA complex could easily be dissociated in lysosomes compared to a low molecular weight complex. Furthermore, the high molecular weight chitosan/DNA complex showed superior transfection efficiency in relation to the low molecular weight complex. Therefore, it could be concluded that the dissociation of the chitosan/DNA complex is a critical event in obtaining the high transfection efficiency of the gene carrier/DNA complex.     

壳聚糖-质粒DNA层层组装及携载DNA量的评价方法

目的 壳聚糖与质粒DNA可以层层组装形成多层膜,可用于金属表面载基因涂层.本研究采用表面等离子共振(SPR)技术,实时检测金属表面与壳聚糖、壳聚糖与质粒DNA(pDNA)的相互作用,并分析壳聚糖携载质粒DNA的能力以及壳聚糖-质粒DNA多层膜在液流作用下的稳定性.方法 在11-巯基十一羧酸处理过的裸金芯片上自组装不同浓度、不同相对分子量的壳聚糖(50～400 ku)和质粒DNA分子.结果 层层组装方法中壳聚糖对质粒的携载量与相对分子量和浓度密切相关,即高浓度、高分子量的壳聚糖可以形成更加厚实的多层膜,并可以携载更多的质粒DNA,自组装形成的多层膜还具有耐液流冲刷性,物理性质稳定.结论 采用壳聚糖与治疗基因的多层自组装可以在血管支架上携载质粒DNA,为探索载基因血管支架涂层的构建提供了新的方法和手段.     

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。     

Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles

The study is focused on the evaluation of the potential bioadhesive behaviour of chitosan and thiolated chitosan (chitosan-TBA)-coated poly(isobutyl cyanoacrylates) (PIBCA) nanoparticles. Nanoparticles were obtained by radical emulsion polymerisation with chitosan of different molecular weight and with different proportions of chitosan/chitosan-TBA. Mucoadhesion was ex vivo evaluated under static conditions by applying nanoparticle suspensions on rat intestinal mucosal surfaces and evaluating the amount of nanoparticles remaining attached to the mucosa after incubation. The analysis of the results obtained demonstrated that the presence of either chitosan or thiolated chitosan on the PIBCA nanoparticle surface clearly enhanced the mucoadhesion behaviour thanks to non-covalent interactions (ionic interaction and hydrogen bonds) with mucus chains. Both, the molecular weight of chitosan and the proportion of chitosan-TBA in the formulation influenced the nanoparticle hydrodynamic diameter and hence their transport through the mucus layer. Improved interpenetration ability with the mucus chain during the attachment process was suggested for the chitosan of high molecular weight, enhancing the bioadhesiveness of the system. The presence of thiol groups on the nanoparticle surface at high concentration (200 x 10(-6) micromol SH/cm2) increased the mucoadhesion capacity of nanoparticles by forming covalent bonds with the cysteine residues of the mucus glycoproteins.     

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.     

Controlling chitosan-based encapsulation for protein and vaccine delivery

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation–coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, were inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters.     

壳聚糖多孔支架制备新方法初探

利用NaCl作为致孔剂,采用干热交联壳聚糖与盐淅沥致孔法制备壳聚糖多孔支架,探索该支架制备方法的可行性和安全性.在研究中利用干热交联壳聚糖与盐淅沥致孔法制备了不同质量比(chitosan/NaCl)的壳聚糖多孔支架和冻干法制备的支架,并对所有支架的孔隙率、孔结构、力学性能等参数指标进行评价.结果表明,利用于热交联壳聚糖与盐淅沥致孔法制备过程中加入致孔剂(NaCl)越多,即NaCl/chitosan质量比越大,所构建多孔支架的平均孔径越大,孔隙率越高,抗拉性则下降.     

Controlling the degradation of covalently cross-linked carboxymethyl chitosan utilizing bimodal molecular weight distribution

Degradability is often a critical property of materials utilized in tissue engineering. Although chitosan, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form scaffolds, its slow and uncontrollable rate of degradation can be an undesirable feature. In this study, we characterize chitosan derivatives formed using a combination of carboxymethylation and a bimodal molecular weight distribution. Specifically, chitosan is carboxymethylated to a theoretical extent of approximately 30% as described in our previous work, in which carboxyl groups possessing negative charges are created at a physiological pH. Carboxymethyl chitosan is used to form films and constructs by varying the ratio of high to low molecular weight (MW) while maintaining the mechanical properties of the polymer. The rate of degradation is found to be dependent upon both the carboxymethylation and the ratio of high to low MW polymer, as determined by dry weight loss in lysozyme solution in PBS. Subsequently, biocompatibility is examined to determine the effects of these modifications upon Neuro-2a cells cultured on these films. Neuro-2a cells adhere and proliferate on the modified films at a comparable rate to those cultured on unmodified films. This data indicates that these chitosan derivatives exhibit tunable degradation rates and result in a promising material system for neural tissue engineering.     

壳聚糖醋酸溶液对凝血作用的研究

研究了不同脱乙酰度和不同分子量壳聚糖醋酸溶液的凝血作用。发现壳聚糖醋酸溶液使抗凝血液中红细胞发生了明显的聚集和变形。通过不同分子量和脱乙酰度壳聚糖的促红细胞聚集实验,证明了低脱乙酰度壳聚糖(60%~70%)使红细胞聚集效果更好,分子量在105~106范围内作用不十分明显。对血液的凝血酶时间(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)的测定结果验证了壳聚糖醋酸溶液凝血机理不依赖于血小板和常规“瀑布”凝血机制。     

Effect of graded levels of dietary nitrite on pulmonary hypertension in broiler chickens and dilatory cardiomyopathy in turkey poults.

In two separate trials, day-old broiler chicks and turkey poults were fed graded levels of dietary sodium nitrite in order to investigate the possible role of nitrite-induced methaemoglobinaemia on the incidence of chicken pulmonary hypertension and spontaneous turkey cardiomyopathy (STC). A transient low level of methaemoglobinaemia was observed after 7 days of exposure to dietary nitrite, but the levels returned to normal afterwards in spite of the presence of nitrite in the diet throughout the experiment. No effect on pulmonary hypertension, as measured by the right ventricle weight to total ventricle weight ratio, was observed in chickens exposed to dietary nitrite. The group of turkey poults receiving 1200 mg/kg nitrite in the diet had a higher incidence of STC than controls (20 v. 5%), although the difference was not statistically significant. The levels of nitrite-induced methaemoglobinaemia were probably too low to be considered as the aetiology of increased STC incidence.     

Using PEG as Progen to Preparate Chitosan Scaffold for Tissue Engineering

Tissue engineering basically made up growing the relevant cell in vitro and extracellular matrix. A major goal of tissue engineering is to preparate porous three dimension scaffold for cell proliferate, migrate, differention and to form the structure of desirable tissue and organ. In this study, the effects of various content and macromolecular weight of PEG to chitosan were investigated and evaluated. The pore morphology of chitosan was controlled by changing the concentration and macromolecular weight of PEG. Chitosan porous scaffold has interconecting porosity. The pore morphology can be controlled with varying PEG concentration and macromolecular weight. The pore size is between 10～50 urn, the degree of swelling in water is 85.70 % .     

Video-gait analysis of functional recovery of nerve repaired with chitosan nerve guides

Quantitative analysis of peripheral nerve regeneration using nerve guides is commonly evaluated through histomorphometry and walking track analysis. We conducted a unique assessment of functional sciatic nerve recovery treated with chitosan nerve guides. We used video-gait analysis to evaluate the extent of functional nerve recovery by measuring the ankle angle at different gait cycle phases. We also correlated the gastrocnemius muscle weight measurements and histological analysis to functional nerve recovery. The chitosan group showed increased functional improvement compared to the control groups at the end of a 12-week period ( p < 0.05). Although both control and chitosan angle measurements were lower than those recorded for presurgery animals, the angle measurements significantly improved over the 12-week period. Stance phase duration of the gait cycle was also recorded, which showed a significant increase over the 12-week time period. The muscle weight parameter indicated a significant decrease in muscle atrophy and restoration of functional strength. Histological analysis revealed that the chitosan nerve guide provided significantly increased axonal growth. The functional results indicated that chitosan nerve guides enhanced functional improvement over no repair processes.     

Microcapsules of alginate-chitosan. II. A study of capsule stability and permeability.

The stability of alginate-chitosan capsules was shown to depend strongly on the amount of chitosan bound to the capsules. When the capsules were made by dropping a solution of sodium alginate into a chitosan solution (one-stage procedure), all the chitosan was located in a thin alginate-chitosan membrane on the surface. These capsules were much weaker than the capsules made by reacting calcium alginate beads in an aqueous solution of chitosan and calcium chloride (two-stage procedure). Capsules with high mechanical strength were obtained after shorter reaction times when the number-average molecular weight of the chitosan was reduced to around 15,000, when the capsules were made more homogeneous and when the capsule diameter was reduced to around 300 microm. When these capsules were treated with calcium sequestrant such as citrate under conditions where calcium alginate gels normally dissolve, they still had a gel core indicating the presence of chitosan throughout the capsule matrix. The permeability of the two-stage capsules was reduced when the chitosan molecular weight was increased and the degree of acetylation was increased, and when the capsules were made more inhomogeneous. The addition of another several layers of alginate and chitosan resulted in capsules virtually impermeable to IgG, suggesting an average capsule pore diameter less than 90 A.     

Electrochemical preparation of chitosan/hydroxyapatite composite coatings on titanium substrates

Composite coatings containing brushite (CaHPO(4). 2H(2)O) and chitosan were prepared by electrochemical deposition. The brushite/chitosan composites were converted to hydroxyapatite/chitosan composites in aqueous solutions of sodium hydroxide. The coatings ranged from approximately 1 to 15% chitosan by weight. Qualitative assessment of the coatings showed adhesion significantly improved over that observed for electrodeposited coatings of pure HA.     

A Study on Dynamics of Drug Release from N-alkyl Chitosan Derivative Membranes In Vitro

INTRODUCTION　　 Among all the controlled-release techniques,the use of membranes is the mostpromising due to theirability to maitain constancy orintelligentresponse in the drugdelivery profiles. Solute transported through hydrogel membranes is generally …     

壳聚糖微球体外降脂活性研究

目的为使壳聚糖能更好地用于减肥降脂,必须提高壳聚糖的降脂活性和降低其毒副作用。方法用喷雾干燥法制备壳聚糖微球,在体外模拟人体消化系统环境考察壳聚糖微球对脂类的结合能力。结果壳聚糖微球粒径范围在2—5μm且用量90mg时对脂类的结合能力达19．53gig,对胆酸的结合能力达27．35mg／g,用量120mg时对游离脂肪酸的结合能力达18．25mg／g,均显著高于普通壳聚糖。结论壳聚糖微球是提高壳聚糖降脂活性的一种有效途径,该研究为新一代壳聚糖减肥类产品的后续开发提供了理论依据。     

Effects of the degree of deacetylation on the physicochemical properties and Schwann cell affinity of chitosan films

Chitosan is a potential material for the preparation of nerve repair conduits. In order to find a better chitosan for the application in peripheral nerve regeneration, the effects of the degree of deacetylation (DD) on the physicochemical properties and Schwann cell affinity of chitosan films have been evaluated. Six kinds of chitosan samples with similar molecular weight, but various DD in a range from 70.1 to 95.6% were prepared from one stock chitosan material and fabricated into films. X-ray diffraction analysis showed that there were more crystalline regions in the higher DD chitosan films. Swelling and mechanical property measurements revealed that the swelling index of chitosan films decreased and their elastic modulus and tensile strength increased with the increase in DD. The adsorption amount of fibronectin and laminin on chitosan films was measured by means of enzyme-linked immunosorbent assay (ELISA). Culture of adult rat Schwann cells on the films showed that the chitosan films with higher DD provided better substrata for Schwann cell spreading and proliferation. In conclusion, DD of chitosan plays an important role in their physicochemical properties and affinity with Schwann cells. The results suggest that chitosan with a DD higher than 90% is considered as a promising material for application in peripheral nerve regeneration.     

Structure, depolymerization, and cytocompatibility evaluation of glycol chitosan

Glycol chitosan, a water soluble chitosan derivative being investigated as a new biomaterial, was fractionated via two different methods. Initial characterization of the glycol chitosan with (1)H NMR spectroscopy illustrated the presence of both secondary and tertiary amine groups, contradictory to its widely accepted structure. Fractionation of glycol chitosan with nitrous acid resulted in a significant reduction in the number average molecular weight, specifically, from 170 to approximately 7 kDa for a pH 3 and below. However, the reaction altered its chemical structure, as the secondary amine groups were converted to N-nitrosamines, which are potentially carcinogenic. An increase in the pH of the reaction limited this formation, but not entirely. Free radical degradation initiated with potassium persulfate was not as effective at reducing the molecular weight as the nitrous acid approach, yielding molecular weights around 12 kDa under the same molar ratio of degrading species, but did retain the structural integrity of the glycol chitosan. Additionally, control of the molecular weight appears feasible with potassium persulfate. When assessed in vitro for cytocompatibility, the polymer exhibited no toxicity on monolayer-cultured chondrocytes, and in fact stimulated cell growth at low concentrations.     

Synthesis,characterization and cytotoxicity of photo-crosslinked maleic chitosan–polyethylene glycol diacrylate hybrid hydrogels

Synthetic hydrogels are important biomaterials for many biomedical applications and hydrogels produced via photo-gelation have shown particular promise. In this paper, we describe a new family of biodegradable hybrid hydrogels fabricated in aqueous solution via long wavelength UV photo-crosslinking using maleic chitosan and polyethylene glycol diacrylate (PEGDA) as precursors. The maleic chitosan precursor was prepared by a simple one-step chemical modification of chitosan, with high yields, and characterized by Fourier transform infrared spectroscopy, ¹H NMR and ¹³C NMR. Maleic chitosan and PEGDA precursors at a wide range of weight feed ratios were mixed in aqueous solution and directly photo-crosslinked for 10 min under a long wavelength UV light (365 nm) using 4-(2-hydroxyethoxy) phenyl-(2-hydroxy-2-propyl) ketone (Irgacure 2959) as photoinitiator. It was observed that as the weight feed ratio of maleic chitosan to PEGDA decreased the pore sizes of the hydrogel samples decreased, thereby increasing the densities of the hydrogel networks and producing a lower swelling ratio and a higher compressive modulus. The molecular weight of PEGDA had a similar effect. Preliminary cell cytotoxicity tests of both the maleic chitosan precursor and maleic chitosan/PEGDA hydrogels, based on the MTT assay and live–dead assay, respectively, showed that these new chitosan-based biodegradable biomaterials were relatively non-toxic to bovine aortic endothelial cells at low dosages.     

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6-8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.