Trypsin has been immobilized by adsorption onto Amberlite XAD-7 beads. The Michaelis constant (Km) of the enzyme was increased about sevenfold following the immobilization. Its rate of penetration into the porous beads was determined by staining the beads, which had been split, with naphthol blue black. The extent of diffusional rate limitation of immobilized trypsin was related to the penetration depth of the enzyme into the beads. This can be controlled by manipulating the conditions during the preparation of the immobilized enzyme.
Aminoacylase I (EC 3.5.1.14) encapsulated in calcium alginate beads stabilized with poly-L-lysine was used for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The immobilized aminoacylase was studied with respect to operational stability, thermal stability, effects of pH and temperature and kinetic constants. The leakage of enzyme from the stabilized beads was eliminated. The immobilized enzyme retained high biological activity. The Km and Vmax values for the stabilized beads were 11.11 mmol dm-3 and 0.076 mumol min-1 respectively. The optimum pH and temperature for the hydrolysis were 6.5 and 55 degrees C respectively. Scanning electron micrographs revealed crosslinked structures on the surface of the beads. The operational performances of the beads in a batch reaction and a packed-bed bioreactor for continuous reaction were investigated. With batch reaction, only about 5% of enzyme activity was lost within ten reaction cycles and there was no significant loss of activity over 600 h of continuous operation after equilibrium was reached, and a conversion yield of about 80% was obtained.
A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40 degrees C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which are affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p-nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non-polar polymers.
This study aimed to determine the role of surfactant protein A (SP-A) in the formation of stable microbubble in tracheal aspirates. Our results showed that as the concentration of anti SP-A antibodies added to tracheal aspirate specimens increased, the number of stable microbubble formed in the specimen decreased. The correlation between stable microbubble counts and the SP-A levels in the tracheal aspirates was good, r = 0.85, p < 0.05. This study suggests that SP-A plays an important role in stable microbubble formation. Measurement of small stable microbubbles is thus a useful bedside test for predicting the SP-A activity in the tracheal aspirates and in indirect measurement of lung maturity.
The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4.3 x 2.0 microns (fresh) and have 15.5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.
Modified-release drug spheroids coated with an aqueous mixture of high-viscosity hydroxypropylmethylcellulose (HPMC) and sodium carboxymethylcellulose (NaCMC) were formulated. The preparation of core drug spheroids and the coating procedures were performed using the rotary processor and a bottom-spray fluidized bed, respectively. Dissolution studies indicated that incorporation of suitable additives, such as poly(vinylpyrrolidone) (PVP) and poly(ethylene glycol) 400 (PEG) improved the flexibility and integrity of the coat layer by retarding the drug release. An increase in coating levels applied generally retarded the release rate of the drug. However, the ratio of HPMC to NaCMC in the mixed, plasticized polymeric coat played a more dominant role in determining the dissolution T50% values. The optimal ratio of HPMC to NaCMC for prolonged drug release was found to be 3:1, whereas an increase in the amount of NaCMC in the mixed polymer coat only increased drug release. The synergistic viscosity effect of HPMC and NaCMC in retarding drug release rate was greater in distilled water than in dissolution media of pH 1 and 7.2. Cross-sectional view of the scanning electron micrograph showed that all of the coated spheroids exhibited a well-fused, continuous, and distinct layer of coating film. The drug release kinetics followed a biexponential first-order kinetic model.
The aim of this study was to investigate the effect of the surfactant properties of polyvinyl alcohol (PVA) in enhancing the yield of small size microspheres. Naltrexone microspheres were prepared by solvent-solvent extraction evaporation process. PVA of various concentrations were added into the aqueous phase prior to the mixing process. The addition of PVA was expected to influence the shape, size distribution, drug loading and drug release profile. The results indicated that it is desirable to increase the weight fraction of the microspheres with size range below 106 mm for the highest possible yield.
The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.
The influence of microwave irradiation on the drug release properties of freshly prepared and aged alginate, alginate-chitosan and chitosan beads was investigated. The beads were prepared by extrusion method with sulphathiazole as a model drug. The dried beads were subjected to microwave irradiation at 80 W for 10 min, 20 min or three consecutive cycles of 10 and 20 min, respectively. The profiles of drug dissolution, drug content, drug stability, drug polymorphism, drug-polymer interaction, polymer crosslinkage and complexation were determined by dissolution testing, drug content assay, differential scanning calorimetry and Fourier transform infra-red spectroscopy. The chemical stability of drug embedded in beads was unaffected by microwave conditions and length of storage time. The release property of drug was mainly governed by the extent of polymer interaction in beads. The aged alginate beads required intermittent cycles of microwave irradiation to induce drug release retarding effect in contrast to their freshly prepared samples. Unlike the alginate beads, the level of polymer interaction was higher in aged alginate-chitosan beads than the corresponding fresh beads. The drug release retarding property of aged alginate-chitosan beads could be significantly enhanced through subjecting the beads to microwave irradiation for 10 min. No further change in drug release from these beads was observed beyond 30 min of microwave irradiation. Unlike beads containing alginate, the rate and extent of drug released from the aged chitosan beads were higher upon treatment by microwave in spite of the higher degree of polymer interaction shown by the latter on prolonged storage. The observation suggested that the response of polymer matrix to microwave irradiation in induction of drug release retarding property was largely affected by the molecular arrangement of the polymer chains.
Hepatocellular carcinoma (HCC) is a malignant tumour of the hepatocyte. It is a common malignancy worldwide and causes almost half a million deaths annually. Asia is a high risk area. Although surgery (hepatectomy or liver transplantation) is the main form of curative treatment, the majority of patients are not eligible for surgery due to extent of tumour and dysfunction of liver. Radiopharmaceuticals used for transarterial treatment of HCC were Yttrium-90 microspheres, Iodine-131 lipiodol, Rhenium-188 lipiodol, and Holmium-166 Chitosan complex. Yittrium-90 microspheres are glass or resin microspheres of mean sphere diameter of 20 to 30 micrometre. The activity administered was about 4 GBq. Reported response rate was about 20%, and median survival was 54 weeks. On inoperable tumours, reported objective response of I-131 lipiodol was 40 to 70%, and median survival was six to nine months. It showed efficacy similar to TACE. In adjuvant treatment following curative resection of HCC, reported three year survival was 86% compared with 46% for the control group. The administered activity in both adjuvant and inoperable HCC was about 2 GBq (55 mCi). Rhenium-188 lipiodol is a new radioconjugate, and using it we treated 70 patients with inoperable HCC. This treatment was a part of a multi-centre trial sponsored by the International Atomic Energy Agency. Partial response was obtained in 17% of cases, while 49% had stable disease at three months, and 34% showed disease progression. In terms of survival, 19% survived one year, 60% for six months, and 90% for three months. The mean activity was about 4.6 GBq (124 mCi). This method was safe and free from adverse effects.
A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.
The role of protein kinase C (PKC) in hydroxyapatite (HA)-induced phagocytosis by RAW 264.7 cells was investigated. The cells were incubated with HA particles at various incubation time and the levels of PKC activity were determined from the cell lysate. To determine the role of PKC, particles were incubated with the cells pretreated with the various concentrations of bisindolylmaleimide, a PKC inhibitor, and phagocytosis was then assessed at 60 min. Latex beads were used as a control. Our results showed that following incubation with HA particles, the levels of PKC activity in RAW264.7 cells was highest at 7 min and then decreased to reach the baseline levels of the controls at 30 min. Pretreatment of the cells with bisindolylmaleimide significantly reduced phagocytosis of HA particles in a dose-dependent pattern. The results of our present study suggest therefore that ingestion of HA by RAW264.7 cells may depend on PKC activity that may act in the early stages of phagocytosis.
The aim of the present work was to investigate the preparation of microspheres as potential drug carriers for proteins, intended for controlled release formulation. The hydrophilic bovine serum albumin was chosen as a model protein to be encapsulated within poly(D,L-lactide-co-glycolide) (50:50) microspheres using a w/o/w double emulsion solvent evaporation method. Different parameters influencing the particle size, entrapment efficiency and in vitro release profiles were investigated. The microspheres prepared with different molecular weight and hydrophilicity of poly(D,L-lactide-co-glycolide) polymers were non porous, smooth surfaced and spherical in structure under scanning electron microscope with a mean particle size ranging from 3.98 to 8.74 mum. The protein loading efficiency varied from 40 to 71% of the theoretical amount incorporated. The in vitro release profile of bovine serum albumin from microspheres presented two phases, initial burst release phase due to the protein adsorbed on the microsphere surface, followed by slower and continuous release phase corresponding to the protein entrapped in polymer matrix. The release rate was fairly constant after an initial burst release. Consequently, these microspheres can be proposed as new controlled release protein delivery system.
AIM: The aim of this study was to prepare insulin-loaded poly(lactic acid)-polyethylene glycol microspheres that could control insulin release at least for 1 week and evaluate their in vivo performance in a streptozotocin-induced diabetic rat model.
METHODS: The microspheres were prepared using a water-in-oil-in-water double emulsion solvent evaporation technique. Different formulation variables influencing the yield, particle size, entrapment efficiency, and in vitro release profiles were investigated. The pharmacokinetic study of optimized formulation was performed with single dose in comparison with multiple dose of Humulin 30/70 as a reference product in streptozotocin-induced diabetic rats.
RESULTS: The optimized formulation of insulin microspheres was nonporous, smooth-surfaced, and spherical in structure under scanning electron microscope with a mean particle size of 3.07 microm and entrapment efficiency of 42.74% of the theoretical amount incorporated. The in vitro insulin release profiles was characterized by a bimodal behavior with an initial burst release because of the insulin adsorbed on the microsphere surface, followed by slower and continuous release corresponding to the insulin entrapped in polymer matrix.
CONCLUSIONS: The optimized formulation and reference were comparable in the extent of absorption. Consequently, these microspheres can be proposed as new controlled parenteral delivery system.
Compaction of controlled-release coated pellets into tablets is challenging because of the fusion of pellets and the rupturing of coated film. The difficulty in compaction intensifies with the use of extremely water-soluble drugs. Therefore, the present study was conducted to prepare and compact pellets containing pseudoephedrine hydrochloride as an extremely water-soluble model drug. The pellets were produced using an extrusion-spheronization technique. The drug-loaded pellets were coated to extend the drug release up to 12-h employing various polymers, and then they were compressed into tablets using microcrystalline cellulose Ceolus KG-801 as a novel tabletting excipient. The in vitro drug release studies of coated pellets and tablets were undertaken using the USP basket method in dissolution test apparatus I. The amount of drug released was analyzed at a wavelength of 215 nm. The combined coatings of hydroxypropyl methylcellulose and Kollicoat SR-30D yielded 12-h extended-release pellets with drug release independent of pH of dissolution medium following zero-order kinetics. The drug release from the tablets prepared using inert Celous KG-801 granules as tabletting excipient was found faster than that of coated pellets. However, a modification in drug release rate occurred with the incorporation of inert Ceolus KG-801 pellets. The drug dissolution profile from tablets containing 40% w/w each of coated pellets and inert granules along with 20% w/w inert pellets was found to be closely similar to that of coated pellets. Furthermore, the friability, tensile strength, and disintegration time of the tablets were within the USP specifications.
Magnetically separable photocatalyst beads containing nano-sized iron oxide in alginate polymer were prepared. This magnetic photocatalyst beads are used in slurry-type reactors. The magnetism of the catalyst arises from the nanostructured particles gamma-Fe(2)O(3), by which the catalyst can be easily recovered by the application of an external magnetic field. These synthesized beads are sunlight-driven photocatalyst. In the system without magnetic photocatalyst beads, no chromium reduction was observed under sunlight irradiation due to the stability of the chromium (VI). Upon the addition of magnetic photocatalyst beads, the photo-reduction of Cr(VI) was completed in just after only 50min under sunlight irradiation due to the photocatalytic activity of the beads. However when placed away from sunlight, the reduction rate of the chromium is just about 10%. These observations were explained in terms of absorption occurrence of chromium (VI) onto the catalyst surface which took place in this reaction. In addition, photo-reduction rate of chromium (VI) was more significant at lower pH. The results suggest that the use of magnetic separable photocatalyst beads is a feasible strategy for eliminating Cr(VI).
Microspheres prepared from rigid guluronic acid- (MG) and flexible mannuronic acid-rich (MC) alginate will undergo different drug release changes with respect to the influence of microwave on the matrix. An in-depth understanding of their differences in drug release changes is attainable through investigating cross-linking agent-free alginate microspheres prepared by spray-drying technique.
A new alcohol oxidase (AOX) enzyme-based formaldehyde biosensor based on acrylic microspheres has been developed. Hydrophobic poly(n-butyl acrylate-N-acryloxy-succinimide) [poly(nBA-NAS)] microspheres, an enzyme immobilization matrix, was synthesized using photopolymerization in an emulsion form. AOX-poly(nBA-NAS) microspheres were deposited on a pH transducer made from a layer of photocured and self-plasticized polyacrylate membrane with an entrapped pH ionophore coated on a Ag/AgCl screen printed electrode (SPE). Oxidation of formaldehyde by the immobilized AOX resulted in the production of protons, which can be determined via the pH transducer. Effects of buffer concentrations, pH and different amount of immobilization matrix towards the biosensor's analytical performance were investigated. The formaldehyde biosensor exhibited a dynamic linear response range to formaldehyde from 0.3-316.2 mM and a sensitivity of 59.41 ± 0.66 mV/decade (R(2) = 0.9776, n = 3). The lower detection limit of the biosensor was 0.3 mM, while reproducibility and repeatability were 3.16% RSD (relative standard deviation) and 1.11% RSD, respectively (n = 3). The use of acrylic microspheres in the potentiometric formaldehyde biosensor improved the biosensor's performance in terms of response time, linear response range and long term stability when compared with thick film immobilization methods.
The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.