Gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres for gadolinium neutron-capture therapy

In order to provide a suitable device that would contain water-soluble drugs, highly water-soluble gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres (CMS-Gd-DTPA) were prepared by the emulsion method using glutaraldehyde as a cross-linker and Span 80 as a surfactant for gadolinium neutron-capture therapy of cancer. The gadolinium content and the mass median diameter of CMS-Gd-DTPA were estimated. The size and morphology of the CMS-Gd-DTPA were strongly influenced by the initial applied weight ratio of Gd-DTPA:chitosan. FTIR spectra showed that the electrostatic interaction between chitosan and Gd-DTPA accelerated the formation of gadolinium-enriched chitosan microspheres. Sufficient amounts of glutaraldehyde and Span 80 were necessary for producing discrete CMS-Gd-DTPA. The CMS-Gd-DTPA having a mass median diameter 11.7microm and 11.6% of gadolinium could be used in Gd-NCT following intratumoral injection.     

Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman

Glutaraldehyde and glyoxal cross-linked microspheres were prepared using chitosan with different molecular weights (MWs) and degrees of deacetylation (DDAs) for sustained release of centchroman under physiological conditions. The DDA in chitosan was determined by different methods, and the samples were categorized as chitosan with low (48%), medium (62%), and high (75%) DDA. The size and shape of the microspheres were determined by scanning electron microscopy (SEM), and hydrophobicity was determined by adsorption of Rose Bengal dye on microspheres cross-linked with glutaraldehyde or glyoxal. The effect of MW, DDA, and degree of cross-linking in microspheres was studied on the degree of swelling, as well as by the loading and release of centchroman. The glyoxal cross-linked microspheres were more compact and hydrophobic and showed better sustained release in companion to chitosan microspheres and glutaraldehyde cross-linked microspheres. The linear fractional release of centchroman with the square root of time indicated a Fickian behavior of centchroman, and the microspheres also showed zero-order release kinetics for centchroman.     

Evaluation of mucoadhesive properties of chitosan microspheres prepared by different methods

The mucoadhesive properties of chitosan microspheres prepared by different method were evaluated by studying the interaction between mucin and microspheres in aqueous solution. The interaction was determined by the measurement of mucin adsorbed on the microspheres. A strong interaction between chitosan microspheres and mucin was detected. The intensity of the interaction was dependent upon the method of preparation of chitosan microspheres and the amount of mucin added. The extent of mucus adsorption was proportional to the absolute values of the positive zeta potential of chitosan microspheres. The zeta potential in turn was found to be dependent upon the method of preparation of microspheres. The adsorption of type III mucin (1% sialic acid content) was interpreted using Freundlich or Langmuir adsorption isotherms. The values ofr ² were greater for Langmuir isotherm as compared with Freundlich isotherm. The adsorption of a suspension of chitosan microspheres in the rat small intestine indicated that chitosan microspheres prepared by tripolyphosphate cross-linking and emulsification ionotropic gelation can be used as an excellent mucoadhesive delivery system. The microspheres prepared by glutaraldehyde and thermal cross-linking showed good stability in HC1 as compared with microspheres prepared by tripolyphosphate and emulsification ionotropic gelation.     

Poly(3-hydroxybutyrate)/chitosan/ketoprofen or piroxicam composite microparticles: Preparation and controlled drug release evaluation

Poly(3-hydroxybutyrate)/chitosan/piroxicam or ketoprofen composite microparticles were prepared by the solid-in-water-in-oil emulsion-solvent evaporation technique with the aim of reducing the burst effect and controlling the drug release. Reservoir-type microparticles, composed of poly(3-hydroxybutyrate) microspheres embedded in a chitosan matrix were prepared. The size and morphological characteristics of the composite microparticles were evaluated in relation to the chitosan concentration and cross-linking with glutaraldehyde. Reservoir-type composite microparticles were obtained using 2.0% and 3.0% w/v chitosan solutions. A significant reduction in the burst effect and prolonged drug release were observed, particularly when higher chitosan and glutaraldehyde concentrations were used.     

尺寸均一的壳聚糖微球的制备及其作为胰岛素控释载体的研究

采用新型微孔膜乳化技术制备了载胰岛素的壳聚糖微球。研究表明,要制备粒径均一的壳聚糖微球,必须将亲水性膜修饰成疏水性;制得的微球粒径和所采用的膜孔径之间存在很好的线性关系,使得微球粒径可控;以胰岛素为模型药物,主要考察了交联剂用量和交联时间对微球表面形态、药物包埋率和微球体外释药特性的影响。结果表明当氨基与醛基的摩尔比为1∶0.7、交联时间为1h时,所得载药微球的包埋率最高,随着戊二醛用量的增加和交联时间的延长,药物体外释放速率减慢。     

壳聚糖装载猪胸腺肽微球给药

目的：以猪胸腺肽为芯材、壳聚糖为壁材,采用乳化交联结合单凝聚法制备猪胸腺肽壳聚糖口服微球。方法：以壁材浓度、交联剂含量、油水比值、芯材壁材比值为四因素设计正交实验,确定微球最佳制备条件,并对其体外释放及稳定性进行研究。结果：制备微球最优化条件为壳聚糖浓度1％、25％戊二醛含量7％、油水比值2：1、壳聚糖与胸腺肽比值1：1;微球在pH1．5的HC1溶液中2h释放30％,在pH6．8及7．4的PBS缓冲液中最终释放度约80％,并在24h达到释放终点;微球30rain突释率约为10％,1h释放率约为20％,其后缓慢而持续地释放;猪胸腺肽壳聚糖微球在0℃保存8个月时微球外观及形态没有差异,药物剩余率约为91．8％。结论：采用乳化交联结合单凝聚法制备的猪胸腺肽壳聚糖口服微球为缓释给药系统的临床应用奠定了理论基础,具有重要的实际应用价值和社会意义。     

用壳聚糖纳米磁性微球纯化血红细胞超氧化物歧化酶

本研究旨在用壳聚糖-聚丙烯酸纳米磁性微球纯化血红细胞超氧化物歧化酶。采用了接枝共聚法,以K2S2O8为引发剂,使壳聚糖(CTS)与聚丙烯酸(PAA)进行自由接枝共聚合成含有两性基团(-NH3,-COOH)的壳聚糖-聚丙烯酸纳米微球。化学共沉淀法制备Fe3O4磁流体,以戊二醛为交联剂,制备壳聚糖-聚丙烯酸纳米磁性微球。用傅里叶变换红外光谱仪对磁性微球结构进行检测。JEM-4000EX电镜技术对微球粒径,形貌进行表征。SOD试剂盒测定各步骤Cu-ZnSOD酶活性。结果表明,壳聚糖-聚丙烯酸纳米磁性微球有较好的粒径分布、磁响应性及蛋白吸附特性。纯化后酶比活性达6 727 U/mg,产品得率21.1%,活性回收85.7%。壳聚糖-聚丙烯酸纳米磁性微球经血液纯化血红细胞SOD具有可再生性、易操作性,其纯化效果取决于金属Cu2+的螯合程度。     

壳聚糖固定化琼脂酶的研究

采用壳聚糖微球对琼脂酶进行固定化,在单因素实验的基础上用正交试验法确定最佳固定化工艺。结果表明:在戊二醛体积分数为2.5%,交联时间为6 h,加酶量为15 mL,固定时间为3 h时固定酶的活力最高;固定化酶的最适反应温度及最适pH分别为50℃和8.5,高于游离酶;同时其热稳定性及操作稳定性均高于游离酶。     

Heavy metal resistant freshwater ciliate, Euplotes mutabilis, isolated from industrial effluents has potential to decontaminate wastewater of toxic metals

The ciliate, Euplotes mutabilis, isolated from industrial wastewater of tanneries of Kasur, Pakistan, showed tolerance against Cd2+ (22 microg ml(-1)), Cr6+ (60 microg ml(-1)), Pb2+ (75 microg ml(-1)) and Cu2+ (22 microg ml(-1)). The heavy metals, Cr and Pb, were randomly selected for determining the capability of the ciliate to reduce the concentration of these metal ions in the medium and to evaluate its potential use as bioremediator of wastewater. The live protozoans could remove 97% of Pb2+ and 98% of Cr6+ from the medium, 96 h after inoculation of the medium containing 10 micro gml(-1) of metal ions. The acid digestion of ciliate showed 89% of Pb2+ and 93% of Cr6+ ions accumulated in the organism. When the ciliate was exposed to heavy metals at a larger scale viz., 10 l of water containing 10 micro gml(-1) of heavy metals, it removed 86% of Pb2+ and 90% of Cr6+ from the medium. The metal uptake ability of E. mutabilis, as evidenced by its survival and growth in 100ml and 10 l of water containing 10 microg ml(-1) of metal ions, reduction in the concentration of heavy metals in the medium and its increased uptake by the live cells, and no metal uptake by the heat killed ciliate can be exploited for metal detoxification of industrial wastes and environmental clean-up operations.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Two-stage optimization process for formulation of chitosan microspheres

The objective of the present study was to optimize the concentration of a chitosan solution, stirring speed, and concentration of drugs having different aqueous solubility for the formulation of chitosan microspheres. Chitosan microspheres (unloaded and drug loaded) were prepared by the chemical denaturation method and were subjected to measurement of morphology, mean particle size, particle size distribution, percentage drug entrapment (PDE), drug loading, and drug release (in vitro). Morphology of the microspheres was dependent on the level of independent process parameters. While mean particle size of unloaded microspheres was found to undergo significant change with each increase in concentration of chitosan solution, the stirring rate was found to have a significant effect only at the lower level (ie, 2000 to 3000 rpm). Of importance, spherical unloaded microspheres were also obtained with a chitosan solution of concentration less than 1 mg/mL. Segregated unloaded microspheres with particle size in the range of 7 to 15 microm and mean particle size of 12.68 microm were obtained in the batch prepared by using a chitosan solution of 2 mg/mL concentration and stirring speed of 3000 rpm. The highest drug load ( microg drug/mg microspheres) was 50.63 and 13.84 for microspheres containing 5-fluorouracil and methotrexate, respectively. While the release of 5-fluorouracil followed Higuchi's square-root model, methotrexate released more slowly with a combination of first-order kinetics and Higuchi's square-root model. The formation of chitosan microspheres is helped by the use of differential stirring. While an increase in the concentration of water-soluble drug may help to increase PDE and drug load over a large concentration range, the effect is limited in case of water-insoluble drugs.     

One-pot synthesis and characterization of cross-linked quaternized chitosan microspheres as protein adsorbent

The one-pot synthesis and characterization of cross-linked quaternized chitosan microspheres (CQCM) as a protein adsorbent are presented. First of all, chitosan particles were prepared by spray drying method, and then they were quaternized and cross-linked in turn with glycidyltrimethylammonium (GTMAC) chloride and glutaraldehyde in isopropanol containing 10% water in one-pot. The effect of the reaction temperature, reaction time and the amounts of added GTMAC and glutaraldehyde on the protein adsorption ability of CQCM was investigated. The adsorption behavior of the CQCM prepared in the optimum synthetic conditions was well described by the Langmuir isotherm with maximum adsorption capacity equal to 1424 mg BSA/g dry weight. The particle size ranged from 7.6 to 48.9 μm. The mechanism of adsorption-desorption of BSA to the CQCM was ion-exchange. Finally, the extraction of soybean peroxidase from crude soybean peroxidase solution using the CQCM was performed.     

Growth kinetics, protease activity and histophagous capability of Uronema sp. infesting cultured silver pomfret Pampus argenteus in Kuwait

Laboratory-produced and -reared sub-adults of silver pomfret (zobaidy) Pampus argenteus affected by severe scuticociliatosis during April and May 2005 were investigated and the causative was a scuticociliate, presumptively identified as Uronema sp. The parasite was capable of producing highly necrotic skin lesions. Mortalities started with a rise in the seawater temperature from 20 to 21.5 degrees C, coinciding with an increase in the total bacterial load of the rearing tank water from 10(3) to 10(5) CFU ml(-1). The parasite was successfully cultured in vitro (85 passages), using 10% brain heart infusion broth inoculated simultaneously with an aquatic Vibrio sp. The parasite reached peak cell density (1.82 x 10(5) cells ml(-1)) on the third day of inoculation. The parasite cell count was found to be inversely related (r = -0.45) to the bacterial count in the incubation medium. The ciliate body dimensions measured, on average, 34.05 +/- 0.99 microm in length and 16.70 +/- 0.74 microm in width (n = 300). Silver staining revealed morphological characteristics of Uronema sp., including a truncated anterior end, a buccal apparatus, caudal cilium and 3 oral polykinetids, with the first one appearing as a single row. Average number of kineties was 12 +/- 3. There was an inverse relationship (R2 = 0.84) between the proteolytic activity and the number of in vitro passages of the scuticociliate. However, the parasite from a freshly infected fish (using inoculum from the tenth passage) showed higher proteolytic activity (31.2%) compared with that produced by the inoculum from in vitro cultures (9.75%). The ciliate also showed a distinct histophagous capability when tested in vitro using host muscle tissue.     

Preparation and characterization of sodium hexameta phosphate cross-linked chitosan microspheres for controlled and sustained delivery of centchroman

The cross-linked microspheres using chitosan with different molecular weights and degree of deacetylation have been prepared in presence of sodium hexameta polyphosphate (SHMP) as physical cross-linker. The degree of cross-linking through electrostatic interactions in chitosan microspheres has been evaluated by varying the charge density on chitosan and varying degree of dissociation of sodium hexameta polyphosphate by solution pH. The degree of deacetylation and molecular weight of chitosan has controlled electrostatic interactions between hexameta polyphosphate anions and chitosan, which played significant role in swelling, loading and release characteristics of chitosan microspheres for centchroman. The microspheres prepared by hexameta polyphosphate anions cross-linker were compact and more hydrophobic than covalently cross-linked microspheres, which has been attributed to the participation of all amino groups of chitosan in physical cross-linking with added hexameta polyphosphate anions. The microspheres prepared under different experimental conditions have shown an initial step of burst release, which was followed by a step of controlled release for centchroman. The extent of drug release in these steps has shown dependence on properties of chitosan and degree of cross-linking between chitosan and added polyanions. The degree of swelling and release characteristics of microspheres was also studied in presence of organic and inorganic salts, which shown significant effect on controlled characteristics of microspheres due to variations in ionic strength of the medium. The initial step of drug release has followed first order kinetics and become zero order after attaining an equilibrium degree of swelling in these microspheres. The microspheres prepared using chitosan with 62% (w/w) degree of deacetylation and molecular weight of 1134 kg mol⁻¹ have shown a sustained release for centchroman for 50 h at 4% (w/w) degree of cross-linking with SHMP.     

Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system.

Chitosan dissolved in acetic acid reacted with glutaraldehyde solution, ranging in concentration from 0.10 to 25.0 x 10(-2) mol dm3. The modified polymers were characterized by means of carbon, hydrogen and nitrogen elemental analysis, scanning electron microscopy, X-ray diffractometry, 13C nuclear magnetic resonance (NMR), infrared and Raman spectroscopies. The uptake of metallic cations in aqueous medium was checked through copper. The obtained data from 13C NMR, infrared and Raman spectroscopies evidenced the formation of an ethylenic double bond in the chitosan glutaraldehyde interaction. These data suggest that free pendant amine groups of chitosan polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds, due to the resonance established with adjacent double ethylenic bonds. The crosslinking is formed by the nonuniform length of chains and by terminal unities. The crosslinking formation can involve two chitosan unities belonging, or not, to the same polymeric chain. The sequence of reactions was established for a chitosan:glutaraldehyde molar proportion of 1:20. The degree of crystallinity and particle size decreased as the amount of glutaraldehyde was increased in the polymer. Physical and chemical properties are not just affected for the chitosan glutaraldehyde reaction, but are also affected strongly by the dissolution of the natural chitosan.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Formulation and evaluation of mucoadhesive glipizide microspheres

The purpose of this research was to formulate and system-atically evaluate in vitro and in vivo performances of mucoadhesive microspheres of glipizide. Glipizide microspheres containing chitosan were prepared by simple emulsification phase separation technique using glutaraldehyde as a cross-linking agent. Results of preliminary trials indicate that volume of cross-linking agent, time for cross-linking, polymer-to-drug ratio, and speed of rotation affected characteristics of microspheres. Microspheres were discrete, spherical, and free flowing. The microspheres exhibited good mucoadhesive property in the in vitro wash-off test and also showed a high percentage drug entrapment efficiency. A 3² full factorial design was employed to study the effect of independent variables, polymer-to-drug ratio (X ₁), and stirring speed (X ₂) on dependent variables percentage mucoadhesion, t₈₀, drug entrapment efficiency, and swelling index. The best batch exhibited a high drug entrapment efficiency of 75% and a swelling index of 1.42; percentage mucoadhesion after 1 hour was 78%. The drug release was also sustained for more than 12 hours. The polymer-to-drug ratio had a more significant effect on the dependent variables. In vivo testing of the mucoadhesive microspheres to albino Wistar rats demonstrated significant hypoglycemic effect of glipizide.     

Evaluation of Ionotropic Cross-Linked Chitosan/Gelatin B Microspheres of Tramadol Hydrochloride

Microspheres of tramadol hydrochloride (TM) for oral delivery were prepared by complex coacervation method without the use of chemical cross-linking agents such as glutaraldehyde to avoid the toxic reactions and other undesirable effects of the chemical cross-linking agents. Alternatively, ionotropic gelation was employed by using sodium-tripolyphosphate as cross-linking agent. Chitosan and gelatin B were used as polymer and copolymer, respectively. All the prepared microspheres were subjected to various physicochemical studies, such as drug–polymer compatibility by thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectroscopy, surface morphology by scanning electron microscopy, frequency distribution, drug entrapment efficiency, in vitro drug release characteristics and release kinetics. The physical state of drug in the microspheres was determined by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). TLC and FTIR studies indicated no drug–polymer incompatibility. All the microspheres showed initial burst release followed by a fickian diffusion mechanism. DSC and XRD analysis indicated that the TM trapped in the microspheres existed in an amorphous or disordered-crystalline status in the polymer matrix. From the preliminary trials, it was observed that it may be possible to formulate TM microspheres by using biodegradable natural polymers such as chitosan and gelatin B to overcome the drawbacks of TM and to increase the patient compliance.     

Effects of waterborne zinc on reproduction, survival and morphometrics of Gyrodactylus turnbulli (Monogenea) on guppies (Poecilia reticulata)

Recent reviews indicate that pollutants in the surrounding macroenvironment directly influence the population dynamics, distribution and dispersal of fish ectoparasites, often leading to increased parasitism. The aim of the current study was to explore the effects of sublethal concentrations of waterborne zinc (up to 240 microg Zn/L) on survival, reproduction and morphometrics of Gyrodactylus turnbulli, a viviparous monogenean infecting the skin and fins of the guppy, Poecilia reticulata. Parasite survival and reproduction on the fish were recorded daily for individual parasites maintained in isolated containers. Both survival and reproduction were reduced in 30 and 120 microg Zn/L, compared with 0, 15, and 60 microg Zn/L indicating direct toxic effects of Zn on the parasite. However, as generation time was unaffected by Zn, we attribute the reduced reproduction to the shorter lifespan. Parasite survival off the fish was monitored hourly. Average lifespan of the detached parasites decreased linearly from 19.5 h in 0 microg Zn/L to 17.3h in 240 microg Zn/L, further supporting the direct toxic effect of Zn to the parasite. In addition, temporal dynamics of parasite morphometrics were monitored from mini-epidemics sampled after 1, 5, 10, and 15 days exposure to various Zn concentrations. All morphological parameters decreased significantly in response both to concentration and duration of exposure to waterborne Zn. Together these data clearly indicate that concentrations as low as 120 microg Zn/L are directly toxic to G. turnbulli.