Synthesis and Characterization of a Hydroxyethyl Derivative of Chitosan and Evaluation of Its Biosafety

Hydroxyethyl chitosan(HE-chitosan) is a water-soluble derivative of chitosan with many apparent biological properties. For example, it is non-toxic and rapidly biodegradable. Moreover, HE-chitosan has advantages in water-solubility, moisture retention and gelling property due to its hydroxyethyl group. However, the biocompatibility and biodegradability of this multifunctional derivative have rarely been documented although they are critical for its application in biomedical and clinical treatments. The purpose of this work was to evaluate the biosafety of HE-chitosan, and draw important clues for its diverse applications. HE-chitosan was synthesized and characterized its chemical structure with FTIR. Its molecular weight(MW) was determined by gel permeation chromatography(GPC), and its deacetylation degree(DD) was investigated through potentiometric analysis. The cytotoxicity of HE-chitosan on mouse fibroblast cell L929 was tested. The biocompatibility and biodegradability of HE-chitosan in rat and rabbit were evaluated. The FTIR results indicated that the hydroxyethyl groups were linked to C6 of chitosan. The GPC analysis confirmed that its Mw was about 90.01 k Da. It was also demonstrated that HE-chitosan had excellent biocompatibility and biodegradability in vivo and had no cytotoxicity on L929. These findings indicated that HE-chitosan can potentially be applied as a biomaterial in tissue engineering, drug delivery, and other biomedical fields.     

Pharmacokinetics and Biodegradation of Chitosan in Rats

Chitosan, an excellent biomedical material, has received a widespread in vivo application. In contrast, its metabolism and distribution once being implanted were less documented. In this study, the pharmacokinetics and biodegradation of fluorescein isothiocyanate(FITC) labeled and muscle implantation administrated chitosan in rats were investigated with fluorescence spectrophotometry, histological assay and gel chromatography. After implantation, chitosan was degraded gradually during its distribution to diverse organs. Among the tested organs, liver and kidney were found to be the first two highest in chitosan content, which was followed by heart, brain and spleen. Urinary excretion was believed to be the major pathway of chitosan elimination, yet 80% of chitosan administered to rats was not trackable in their urine. This indicated that the majority of chitosan was degraded in tissues. In average, the molecular weight of the degradation products of chitosan in diverse organs and urine was found to be 65 k Da. This further confirmed the in vivo degradation of chitosan. Our findings provided new evidences for the intensive and safe application of chitosan as a biomedical material.     

Establishing Gene Delivery Systems Based on Small-Sized Chitosan Nanoparticles

Chitosan is a natural cationic polysaccharide,which is often used for preparing biomedical materials because of its high biocompatibility.In this study,chitosan with a molecular weight of 160 k Da was chosen to prepare chitosan nanoparticles(CSNPs) as gene vectors by ionic cross-linking with tripolyphosphate(TPP).CSNPs were characterized in terms of particle size,zeta potential,and polydispersity index(PDI) using a Zetasizer,and morphology was evaluated by transmission electron microscopy(TEM).Furthermore,the cytotoxicity and biocompatibility of CSNPs were correspondingly examined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and histological examination.Agarose gel electrophoresis and UV spectrophotometric methods were performed to measure the loading capacity.The cell transfection efficiency of CSNPs loaded with plasmids or si RNA was analyzed by fluorescence microscopy or laser scanning confocal microscopy.The results showed that CSNPs were prepared successfully by the ionic gelation method,which had a smaller particle size(100 nm-200 nm),stable dispersibility,low cytotoxicity,good tissue-biocompatibility,and high gene-loading efficiency.These CSNPs could transfer the plasmids or si RNA to cells.However,CSNPs might have a much higher transfection efficiency for si RNAs than for plasmids,which implies that CSNPs might be a safer and more efficient vector for delivering si RNAs rather than plasmids.     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Analgesis and wound healing effect of chitosan and carboxymethyl chitosan on scalded rats

Analgesis and wound healing effect of chitosan and carboxymethyl chitosan on scalded rats were investigated. A II degree scald model was established in rats, which was subsequently treated with chitosan and carboxymethyl chitosan solution, respectively. The concentration of bradykinin and 5-hydroxytryptophan was detected by assaying enzyme-linked immunosorbent. Healing condition was observed and pathological sections were made to determine the healing effect of chitosan and carboxymethyl chitosan. Results showed that the concentration of bradykinin and 5-hydroxytryptophan peaked at the third hour post-wound in all groups, while the concentration of hydroxyproline peaked at the seventh day post-wound in both chitosan and carboxymethyl chitosan group. The concentration of bradykinin and 5-hydroxytryptophan of carboxymethyl chitosan group was significantly lower than that of control (P < 0.05), while that of chitosan group was similar to that of control (P > 0.05). These findings indicated that carboxymethyl chitosan reduced the concentration of algogenic substances, resulting in analgesia. During the whole recovery process, the hydroxyproline concentration in chitosan and carboxymethyl chitosan group on day 3 and 7 was significantly higher than that of control (P < 0.01); however the significance of such a highness decreased on day 14 (P < 0.05). These findings indicated that chitosan and carboxymethyl chitosan accelerated tissue repair. Meanwhile, chitosan performed better in healing than carboxymethyl chitosan in both decrustation and healing time. In conclusion, carboxymethyl chitosan showed significant analgesis and wound-healing promotion effect, but chitosan only showed wound-healing promotion effect.     

Effects of sulfate chitosan derivatives on nonalcoholic fatty liver disease

Sulfate chitosan derivatives have good solubility and therapeutic effect on the cell model of NAFLD. The aim of this study was to examine the therapeutic effect of sulfate chitosan derivatives on NAFLD. The male Wistar rats were orally fed high fat emulsion and received sulfate chitosan derivatives for 5 weeks to determine the pre-treatment effect of sulfate chitosan derivatives on NAFLD. To evaluate the therapeutic effect of sulfate chitosan derivatives on NAFLD, the rats were orally fed with high concentration emulsion for 5 weeks, followed by sulfate chitosan derivatives for 3 weeks. Histological analysis and biomedical assays showed that sulfate chitosan derivatives can dramatically prevent the development of hepatic steatosis in hepatocyte cells. In animal studies, pre-treatment and treatment with sulfate chitosan derivatives significantly protected against hepatic steatohepatitis induced by high fat diet according to histological analysis. Furthermore, increased TC, ALT, MDA, and LEP in NAFLD were significantly ameliorated by pre-treatment and treatment with sulfate chitosan derivatives. Furthermore, increased TG, AST, and TNF-α in NAFLD were significantly ameliorated by treatment with sulfate chitosan derivatives. Sulfate chitosan derivatives have good pre-treatment and therapeutic effect on NAFLD.     

SYNTHESIS AND PROPERTIES OF SULFHYDRYL CHITOSAN

A new adsorbent for heavy metal ions, sulfhydryl chitosan (S-chitosan), was produced by treatment of chitosan with sulhydryl acetic acid in the presence of sulfuric acid as a catalyst. Its structure was confrrmed by elemental analysis and FI‘-IR spectra analysis. The adsorption properties of sulthydryl chitosan for Cu(Ⅱ ), Cd(Ⅱ ), Pb(Ⅱ), Cr(Ⅲ) and Ni(Ⅱ) were investigated, and the effect of pH value on adsorption, adsorption kinetics, and selective adsorption was examined. It was shown that S-chitosan has good adsorption for Pb(Ⅱ), Cu(Ⅱ) and Cd(Ⅱ) like chitosan, is also insoluble in acid solution; has good adsorption kinetic properties for heavy metal ions; and can be used in acid solution. The adsorption capacities of S-chitosan can be affected by media acidity. The adsorbed Cu(Ⅱ) Cd(Ⅱ) and Pb(Ⅱ) could be eluted by diluted chlorhydric acid.     

Adsorption of heavy metal ions, dyes and proteins by chitosan composites and derivatives — A review

Chitosan composites and derivatives have gained wide attentions as effective biosorbents due to their low costs and high contents of amino and hydroxyl functional groups. They have showed significant potentials of removing metal ions, dyes and proteins from various media. Chemical modifications that lead to the formation of the chitosan derivatives and chitosan composites have been extensively studied and widely reported in literatures. The aims of this review were to summarize the important information of the bioactivities of chitosan, highlight the various preparation methods of chitosan-based active biosorbents, and outline its potential applications in the adsorption of heavy metal ions, dyes and proteins from wastewater and aqueous solutions.     

Researches on the Internal Molecular Weight Uniformity of Chitosan Biomaterials

Uniform molecular weight(Mw)chitosan(CS)is highly demanded in medical biomaterial industry.This present article described heterogeneous degradation of CS in aqueous HCl/ethanol solution,in which progress uniform Mw CS was successfully prepared.The Mw distribution of CS was measured by gel permeation chromatography(GPC)analysis.Moreover,the structure and properties of degraded CS were characterized by Fourier transform infrared spectroscopy(FT-IR),nuclear magnetic resonance spectroscopy(^1H NMR),X-ray diffraction(XRD)and thermogravimetric(TG)analysis.In addition,the biocompatibility of degraded CS was also assessed by hemolysis rate(HR)measurement.The Mw of CS dramatically decreased from 246 KDa to 76 k Da at the initial 30 min,and stabilized at 18 kDa after 24 h.GPC analysis results showed that the degraded CS molecular become homogenization.FT-IR and 1 H NMR analysis confirmed the basic structure of CS molecular backbone was not destroyed during this progress.Besides,the water solubility of CS was not significantly influenced by this reaction.Moreover,the XRD analysis revealed that crystallinity of degraded CS increased from 70.32% to 99.25%with time.The TG analysis showed improved thermal stability of degraded CS.HR measurement demonstrated the degraded CS possessed excellent biocompatibility.This simple and efficient heterogeneous degradation would open up a new route to produce uniform Mw CS.     

恩诺沙星在凡纳滨对虾体内的代谢和残留消除规律

在26±2℃水温下,每天投喂含有恩诺沙星药物的饲料,研究恩诺沙星在凡纳滨对虾Litopenaeus vanname肌肉、肠和肝胰脏组织中的代谢和残留消除规律。残留药物用乙腈提取,液相色谱串联质谱仪检测。结果表明:恩诺沙星在凡纳滨对虾体内可代谢为环丙沙星,对虾体内同时有恩诺沙星和环丙沙星两种药物残留;环丙沙星在肌肉、肝胰脏和肠组织中的消除时间分别为6、8、10 d,而恩诺沙星在这三组织中的消除时间则为12、14、16 d。建议把肠作为该药残留监控的靶组织,凡纳滨对虾的休药期不少于16 d。     

Preparation and characterization of magnetic resin made from chitosan and cerium

In this study,the water-based ferromagnetic fluid and magnetic resin made from chitosan and cerium complex(MRCCC) were successfully prepared by using the chemical co-precipitation technique and by the reversed-phase suspension cross-linking polymerization.MRCCC presented uniform and narrow particle size distribution as determined by the Laser Particles Sizer.The Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES),Fourier transform infrared spectroscopy(FT-IR),differential scanning calorimetry(DSC)and X-ray powder diffraction(XRD)study demonstrated that there were iron and cerium existing in MRCCC.The movement of MRCCC under magnetic field proved its magnetic property.The swelling kinetics in water or solutions with different pH indicated that MRCCC could be applied in solutions with pH greater than 1.0.The ferromagnetic fluid particles were stable in MRCCC soaked in solutions with pH >2.0.In view of these results,MRCCC can be used as material for separation,clarification,adsorption,sustained release and hydrolysis activity.     

复方新诺明在罗氏沼虾中的药代动力学和组织分布

研究了单剂量肌肉注射给药[甲氧苄氨嘧啶(SMZ)0 60 mg/kg]、口灌给药[磺胺甲基异噁唑(SMZ)100 mg/kg]和复方口灌给药(SMZ 100 mg/kg和TMP 20 mg/kg)三种给药方式下,SMZ和TMP在罗氏沼虾中的代谢动力学,分析了SMZ和TMP血淋巴、肌肉和肝胰腺中的分布特征。结果表明,SMZ以肌肉注射方式给药后的血淋巴中,药物经时浓度曲线符合二室开放房室模型,而SMZ&TMP口灌给药后却不能用房室模型拟合。肌注给药后,SMZ的消除半衰期(t1/2β)为24.92 h,单方和复方口灌给药后SMZ的消除半衰期(t1/2β)分别为40.70和47.197 h。复方口灌给药后TMP的消除半衰期(t1/2β)为28.77 h,TMP对SMZ的药代动力学并无显著影响。口灌给药后,SMZ和TMP在肝胰腺中的药物浓度高于同时间肌肉中的药物浓度,但肌肉和肝胰腺中的SMZ消除速度基本一致。     

Establishment of a spleen cell line from large yellow croaker Pseudosciaena crocea and its primitive application in foreign gene transfection

A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco’s modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.     

Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber (Apostichopus japonicus)

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B(Bf) serves as the catalytic subunit of complement component 3(C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber(Apostichopus japonicus), termed Aj Bf, was obtained by rapid amplification of c DNA ends(RACE). The full-length c DNA of Aj Bf was 3231 bp in length barring the poly(A) tail. It contained an open reading frame(ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR(5'-terminal untranslated region) and a 384 bp 3'-UTR. Aj Bf was a mosaic protein with six CCP(complement control protein) domains, a VWA(von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of Aj Bf protein was 101 k Da. Quantitative real time PCR(q RT-PCR) analysis indicated that the expression level of Aj Bf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that Aj Bf expressed higher in coelomocytes than in other four tissues. In addation, Aj Bf expression in different tissues was induced significantly after LPS or Poly I:C challenge. These results indicated that Aj Bf plays an important role in immune responses to pathogen infection.     

Localization and Dynamic Expression of a 27.8kDa Receptor Protein for Lymphocystis Disease Virus Infection in Sea Bass(Lateolabrax japonicus) Tissues

Lymphocystis disease virus(LCDV) infects target cells by attaching to a 27.8 k Da receptor(27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies(MAbs) have been developed. However, the 27.8R existence in tissues of sea bass(Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

壳聚糖对腐生葡萄球菌的作用机制

【目的】探究壳聚糖处理对腐生葡萄球菌(Staphylococcus saprophytic)的作用机制。【方法】通过光密度值结合菌液澄清度法综合确定壳聚糖对腐生葡萄球菌的最小抑菌浓度(Minimum Inhibitory Concentration,MIC),分析其在MIC与2MIC的壳聚糖作用下的微生物生长曲线、电导率值、碱性磷酸酶(Alkaline Phosphatase,AKP)含量、过氧化氢酶(Catalase,CAT)含量、苹果酸脱氢酶(Malatedehydrogenase,MDH)含量与生物膜生成量等指标变化,并结合扫描电镜(ScanningElectronMicroscopy,SEM)观察其作用后对菌体细胞形态变化影响,综合评价壳聚糖处理对腐生葡萄球菌的作用机制。【结果与结论】壳聚糖对腐生葡萄球菌的MIC为1.25 mg/mL;菌体经MIC和2MIC处理后,其正常生长受到抑制,细胞壁被破坏,细胞膜通透性改变,CAT与MDH含量降低,其作用效果与壳聚糖浓度正相关;SEM结果显示,与对照CK组对比,腐生葡萄球菌经MIC壳聚糖处理后,菌体形状不规则,表面皱褶;经2MIC壳聚糖处理后,菌体表面产生泡状物且黏附明显,表明壳聚糖使腐生葡萄球菌菌体细胞壁受损,菌体生长受到抑制,胞内保护酶与三羧酸循环中的关键酶活性降低,细胞代谢能力随之下降,细胞膜结构完整度受到破坏,最终导致菌体死亡。     

Dietary Supplementation with Sea Cucumber Saponins and Exercise Can Significantly Suppress Adipose Accumulation in Mice Fed with High-Fat Diet

Dietary supplementation with sea cucumber saponins(SCS) and exercise have been confirmed to be effective in preventing the development of obesity and its related diseases. However, the combined effectiveness of these interventions has not been explored. Here, we studied whether the beneficial influences of exercise could be further enhanced by dietary supplementation with SCS in high-fat diet-fed KM(Kunming) mice. Mice were randomly divided into four groups, including the high-fat diet group(HF), the SCS group(HF-S), the exercise group(HF-E), and the combination of dietary SCS and exercise group(HF-S + E). There were eight mice in every group. The results demonstrated that the combination of dietary SCS and exercise could synergistically reduce fat accumulation. In particular, white adipose tissue decreased by 63% in the HF-S + E group compared with that in the HF group. SCS supplementation with exercise also improved peripheral markers, such as serum parameters and hepatic TG levels. Further mechanical testing indicated that the combined effects of dietary SCS and exercise on inhibiting fat accumulation might be attributed to the inhibition of lipid synthesis in the liver and the activation of lipolysis in white adipose tissue to increase energy consumption.     

The aggregation behavior and formation of nanoparticles of oleoylchitosan in dilute aqueous solution

Oleoylchitosans (O-chitosans) with different degrees of substitution (DS) were synthesized by reacting chitosan with oleoyl chloride. The chemical structures of the products were characterized by 1H NMR and FT-IR. These results suggested the for-mation of an amide linkage between the amino groups of chitosan and the carboxyl groups of oleic acid. The viscosity of O-chitosan sharply increased with the increase of concentration, whereas that of unmodified chitosan rose only slightly. This increase became larger as the DS increased. All of the O-chitosans could reduce surface tension slightly. The critical aggregation concentration (CAC) of O-chitosans with DS 5%, 11%, and 27% were 79.43 mgL-1, 31.6 mgL-1, and 10mgL-1, respectively. Nanoparticles were prepared using an O/W emulsification method. The mean diameters of the polymeric amphiphilic nanoparticles of O-chitosans with DS 5% and 11% were around 327.4 nm and 275.3 nm, respectively.     

Amphiphilic sodium alginate-vinyl acetate microparticles for drug delivery

To overcome the fast or burst release of hydrophilic drugs from hydrophilic alginate-based carriers, hydrophobic molecule(vinyl acetate, VAc) was grafted on alginate(Alg), which was further used to prepare drug carriers. Amphiphilic Alg-g-PVAc hydrogel beads were firstly prepared by emulsification/internal gelation technique for the loading of bovine serum albumin(BSA). Then, chitosan was coated on the surface of beads to form novel amphiphilic Alg-g-PVAc/chitosan(Alg-g-PVAc/CS) microcapsules.The BSA-loading amphiphilic Alg-g-PVAc/chitosan(Alg-g-PVAc/CS) microcapsules display similar morphology and size to the hydrophilic alginate/chitosan(AC) microcapsules. However, the drug loading and loading efficiency of BSA in Alg-g-PVAc/CS microcapsules are higher, and the release rate of BSA from Alg-g-PVAc/CS microcapsules is slower. The results demonstrate that the introduction of hydrophobic PVAc on alginate can effectively help retard the release of BSA, and the higher degree of substitution is,the slower the release rate is. In addition, the complex membrane can also be adjusted to delay the release of BSA. As a whole, amphiphilic sodium alginate-vinyl acetate/CS microparticles could be developed for macromolecular drug delivery.