An open tubular capillary electrochromatography column with porous inner surface for protein separation

An open tubular capillary electrochromatography (OTCEC) column using sole porogen to form porous inner surface has been developed. The porous layer was coated on the capillary inner wall by in situ polymerization in the presence of porogen. The results show that the columns using 1-propanol as sole porogen are appropriate for protein separation. It has higher separation efficiency than the column with the usual coporogen due to much more micropores and mesopores on the porous surface and a higher specific surface area. In addition, the sensitivity of the prepared OTCEC column was improved greatly compared with the dynamically coated capillary with polyvinylpyrrolidone.     

Formation of nano-hydroxyapatite in gelatin droplets and the resulting porous composite microspheres

A one-pot strategy was first presented in this paper to synthesize gelatin/hydroxyapatite (HAP) composite microspheres in a water-in-oil (W/O) emulsion. Using gelatin droplets as microreactors and colloid protective medium, needle-like nano-HAP crystals (5 nm x 60-100 nm) in form of clusters were homogeneously and orderly precipitated within gelatin matrix. The results of scanning electron microscopy (SEM) revealed that the as-prepared microspheres with an average diameter of 7.5 microm displayed a narrow particle size distribution, a high dispersity and a naturally porous structure. This microsphere material is expected to have a great potential for both controlled drug release and faster bone in-growth in bone tissue engineering.     

Corn porous starch: preparation, characterization and adsorption property

This study was carried out to develop a new type of modified starch based on α-amylase and glucoamylase. The structural and chemical characteristics of the porous starch were determined by Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). The potential application of the porous starch as an adsorbent was evaluated using methyl violet as an adsorbed model. The adsorption capacity was optimized by investigating the reaction factors, including the mass ratio of α-amylase to glucoamylase (m_α-amylase/m_glucoamylase), the mass ratio of total amount of enzymes to starch (m_enzyme/m_St), the ratio of liquid volume to starch mass (VH₂O/mSt), pH value of the reaction solution, enzymatic reaction temperature, and enzymatic reaction time. The hydrolysis ratio of each sample was also determined to investigate the effect of different reaction conditions on the hydrolysis degree. The results suggest that the porous starch has a more excellent adsorption capacity than the native starch, and may be expected to have wide potential applications in many fields.     

Preparation of biodegradable microspheres and matrix devices containing naltrexone

In this study, the use of biodegradable polymers for microencapsulation of naltrexone using solvent evaporation technique is investigated. The use of naltrexone microspheres for the preparation of matrix devices is also studied. For this purpose, poly(L-lactide) (PLA) microspheres containing naltrexone prepared by solvent evaporation technique were compressed at temperatures above the Tg of the polymer. The effect of different process parameters, such as drug/polymer ratio and stirring rate during preparation of microspheres, on the morphology, size distribution, and in vitro drug release of microspheres was studied. As expected, stirring rate influenced particle size distribution of microspheres and hence drug release profiles. By increasing the stirring speed from 400 to 1200 rpm, the mean diameter of microspheres decreased from 251 μm to 104 μm. The drug release rate from smaller microspheres was faster than from larger microspheres. However, drug release from microspheres with low drug content (20% wt/wt) was not affected by the particle size of microspheres. Increasing the drug content of microspheres from 20% to 50% wt/wt led to significantly faster drug release from microspheres. It was also shown that drug release from matrix devices prepared by compression of naltrexone microspheres is much slower than that of microspheres. No burst release was observed with matrix devices. Applying higher compression force, when compressing microspheres to produce tablets, resulted in lower drug release from matrix devices. The results suggest that by regulating different variables, desired release profiles of naltrexone can be achieved using a PLA microparticulate system or matrix devices.     

Evaluation of porous carrier-based floating orlistat microspheres for gastric delivery

The purpose of this research was to prepare floating microspheres consisting of (1) calcium silicate as porous carrier; (2) orlistat, an oral anti-obesity agent; and (3) Eudragit S as polymer, by solvent evaporation method and to evaluate their gastro-retentive and controlled-release properties. The effect of various formulation and process variables on the particle morphology, micromeritic properties, in vitro floating behavior, percentage drug entrapment, and in vitro drug release was studied. The gamma scintigraphy of the optimized formulation was performed in albino rabbits to monitor the transit of floating microspheres in the gastrointestinal tract. The orlistat-loaded optimized formulation was orally administered to albino rabbits, and blood samples collected were used to determine pharmacokinetic parameters of orlistat from floating microspheres. The microspheres were found to be regular in sphae and highly porous. Microsphere formulation CS4, containing 200 mg calcium silicate, showed the best floating ability (88%±4% buoyancy) in simulated gastric fluid as compared with other formulations. Release pattern of orlistat in simulated gastric fluid from all floating microspheres followed Higuchi matrix model and Peppas-Korsmeyer model. Prolonged gastric residence time of over 6 hours was achieved in all rabbits for calcium silicate-based floating microspheres of orlistat. The enhanced elimination half-life observed after pharmacokinetic investigations in the present study is due to the floating nature of the designed formulations.     

Preparation, characterization, and in vivo evaluation of salmon calcitonin microspheres

Purpose. This study was done to prepare, characterize, and evaluate salmon calcitonin (sCT) microspheres (ms) in vivo using a low molecular weight, hydrophilic 50∶50 poly (D,L-lactide-co-glycolide) polymer (PLGA).Methods. sCT ms were prepared by a dispersion/solvent extraction/evaporation process and characterized for drug content, particle size, surface morphology, and structural integrity of encapsulated peptide. Peptide stability and binding to the polymer was studied in 0.1 M phosphate buffer (PB), pH 7.4, and 0.1 M acetate buffer (AB), pH 4.0. Serum sCT levels were monitored for 2 weeks after subcutaneous injection of sCT ms to rats.Results. sCT ms were essentially free of discernible surface pores with a particle size distribution in the range of 16 to 89 mm and mean particle size of 51 and 53 mm for 2 batches. Fourier Transform Matrix-assisted Laser Desorption mass spectrometry of the extracted peptide showed that the encapsulation process did not alter its chemical structure. The peptide was substantially more stable in AB than in PB. Peptide binding to the polymer was dependent on pH and was markedly higher in PB than in AB. In vivo study proved that elevated serum sCT levels could be sustained for at least 10 days after administration of sCT ms to rats at a dose of 1.0 mg/kg.Conclusions. It was demonstrated that sCT could be incorporated into polymeric ms prepared from a low molecular weight, hydrophilic PLGA using a dispersion technique without altering molecular structure. A 2-week formulation was prepared at a dose of 1.0 mg/kg.     

氧化葡萄糖杆菌合成右旋糖酐糊精酶的pH两阶段控制策略

为了进一步提高氧化葡萄糖杆菌右旋糖酐糊精酶的产量,在3 L发酵罐水平上考察了pH(3.5?6.0)对菌体生长和产酶的影响。基于不同pH发酵过程中菌体生长及产物合成的变化,确定了pH两阶段控制策略,即0?6 h时控制pH 5.0,6 h后将pH调至4.0。通过采用这一优化策略,右旋糖酐糊精酶酶活有了较大的提高,可达4.03 U/mL,比不控制pH模式下提高了38.5%,是摇瓶水平的12.5倍,同时发酵时间从47 h缩短为15 h。     

多微孔胺基树脂的制备及其对胆红素的吸附性能

采用悬浮聚合法制备了一定尺寸的多微孔聚苯乙烯(PS)微球,然后通过Friedel-Crafts反应(用氯乙酰氯替代了有致癌性的氯甲醚)和胺化反应得到新型的胺基树脂,并对反应条件进行了优化。结果表明,利用最优化条件制备的胺基树脂,其离子交换容量为4.1587mmol/g。考察了新型树脂对胆红素的吸附性能,其吸附量最大可达30.85mg/g,吸附率可达80%。     

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: a comparative raman microscopy study

Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering.     

Spray-dried mucoadhesive microspheres: Preparation and transport through nasal cell monolayer

The purpose of this research was to prepare spray-dried mucoadhesive microspheres for nasal delivery. Microspheres composed of hydroxypropyl methylcellulose (H), chitosan (CS), carbopol 934P (CP) and various combinations of these mucoadhesive polymers, and maltodextrin (M), colloidal silicon dioxide (A), and propylene glycol (P) as filler and shaper, were prepared by spray-drying technique. Using propranolol HCl as a model drug, microspheres were prepared at loadings exceedings 80% and yields between 24% and 74%. Bulky, free flowing microspheres that had median particle size between 15 and 23 μm were obtained. Their zeta potential was according to the charge of polymer. Adhesion time of mucoadhesive microspheres on isolated pig intestine was ranked, CS>CP: H>CP>H, while the rank order of swelling was CP>CS>H. Increasing the amount of CP in CP∶H formulations increased the percentage of swelling. Infrared (IR) spectra showed no interaction between excipients used except CS with acetic acid. The release of drug from CP and CP∶H microspheres was slower than the release from H and CS microspheres, correlated to their viscosity and swelling. Long lag time from the CP microspheres could be shortened when combined with H. The permeation of drug through nasal cell monolayer corresponded to their release profiles. These microspheres affected the integrity of tight junctions, relative to their swelling and charge of polymer. Cell viability was not affected except from CS microspheres, but recovery could be obtained. In conclusion, spray-dried microspheres of H, CS, CP, and CP∶H could be prepared to deliver drug through nasal cell monolayer via the opening of tight junction without cell damaging. Published: February 10, 2006     

Fabrication and characterization of rigid magnetic monodisperse microspheres for protein adsorption

This article describes the fabrication of a rigid magnetic monodisperse bead (M-PGMA-TRI, 4.92 microm) with polyglycidyl methacrylate (PGMA) cross-linked by trimethylolpropane trimethacrylate (TRI). This was realized by adding a proper amount (2%, w/w) of TRI after 3 h of the dispersion-polymerization reaction with the monomer of GMA. The mono-sized microspheres were further processed to introduce magnetic granules by sulfonation and penetration-deposition approaches. The monodisperse bead (M-PGMA) without TRI addition was also fabricated for comparison. The morphology, size and magnetic characteristics of the microspheres were extensively characterized. The M-PGMA-TRI microspheres were nonporous, of smooth surface and superparamagnetic with a saturation magnetization of 13.0 emicro/g. Recycled use of the material for protein adsorption exhibited stability of the magnetic properties of the M-PGMA-TRI, as compared to the significant loss of the saturation magnetization of the M-PGMA. The chemical stability of the M-PGMA-TRI was also confirmed by examining its protein adsorption and magnetic properties after incubation in various solutions such as acidic buffer (pH 2.2) for 24 h. The adsorption capacity of gamma-globulin reached 287.2 mg/g and kept stable in the repeated adsorption/desorption/regeneration cycles. The results indicated that the introduction of 2% TRI was promising for producing rigid magnetic mono-sized microspheres for protein adsorption.     

Application of microcolumn ion chromatography using anion exchangers modified with dextran sulfate for the determination of alkali and alkaline-earth metal ions

Microcolumn ion chromatography using anion exchangers modified with dextran sulfate has been applied to the determination of alkali and alkaline-earth metal ions contained in guinea pig serum and bovine serum. These serums contained Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ and they were indirectly detected at 200 nm. The determination was done without any pretreatment procedure other than dilution.     

Development of rigid bidisperse porous microspheres for high-speed protein chromatography

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.     

Preparation,in vitro release,in vivo absorption and biocompatibility studies of insulin-loaded microspheres in rabbits

The purpose of this study was to develop a single-dose insulin delivery system based on poly (lactide-co-glycolide) (PLGA) microspheres to provide basal insulin level for a prolonged period. Insulin-loaded PLGA microspheres were prepared by water-in-oil-in-water double emulsion (batch A) and solid-in-oil-in-water emulsion (batch B) methods. Microspheres were characterized for physical characteristics and in vitro release. In vivo absorption of insulin and biocompatibility of insulin-loaded PLGA microspheres were performed in diabetic New Zealand white rabbits. Light and transmission electron microscopy were performed on the skin tissues excised from microspheres injected sites in order to study the biocompatibility. The burst release of insulin was high (47%) from batch B and low (5%) from batch A. Therefore, we mixed microspheres of batch A and B in ratio of 3:1 w/w, which produced desirable in vitro release profile. In vivo absorption study showed that insulin-loaded microspheres provided a serum insulin level of 20-40 microU/ml up to 40 days. Biocompatibility study provided evidence of normal inflammatory and foreign body reactions, which were characterized by the presence of macrophages, fibroblasts and foreign body giant cells. Neither necrosis nor tissue damage was identified. At the end of 12 weeks, no distinct histological differences were observed in comparison to the control tissue samples. In conclusion, insulin-loaded PLGA microspheres controlled the in vivo absorption of insulin to maintain the basal insulin level for longer period and the delivery system was biocompatible.     

Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

A novel magnetic poly(vinyl acetate (VAc)–divinyl benzene (DVB)) material (8–34 μm) was synthesized by copolymerization of vinyl acetate and divinyl benzene using oleic acid-stabilized magnetic colloids as magnetic cores. The magnetic colloids and the copolymer microspheres were characterized with transmission and scanning electron microscopes, respectively. Magnetization of the microspheres could be described by the Langevin function. All the observations indicated that the microspheres were superparamagnetic. Magnetic sedimentation of the microspheres was achieved within 3 min, over 300 times faster than the gravitational sedimentation. Candida cylindracea lipase (CCL) was immobilized to the porous carrier at up to 6750 IU/g carrier, remarkably higher than the previous studies. The pH and temperature dependencies of the immobilized CCL were investigated and the optimum temperature and pH for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after recovered by magnetic separations.     

Dextransucrase and the mechanism for dextran biosynthesis

Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.; Fabre, E.; Potocki-Veronese, G.; Monsan, P.; Remaud-Simeon, M. J. Biol. Chem., 2006, 281, 31254-31267] have recently proposed that a truncated Escherichia coli recombinant B-512F dextransucrase uses sucrose and the hydrolysis product of sucrose, d-glucose, as initiator primers for the nonreducing-end synthesis of dextran. Using ¹⁴C-labeled d-glucose in a dextransucrase-sucrose digest, it was found that <0.02% of the d-glucose appears in a dextran of M_n 84,420, showing that d-glucose is not an initiator primer, and when the dextran was treated with 0.01 M HCl at 80 °C for 90 min and a separate sample with invertase at 50 °C for 24 h, no d-fructose was formed, indicating that sucrose is not present at the reducing-end of dextran, showing that sucrose also was not an initiator primer. It is further shown that both d-glucose and dextran are covalently attached to B-512FMC dextransucrase at the active site during polymerization. A pulse reaction with [¹⁴C]-sucrose and a chase reaction with nonlabeled sucrose, followed by dextran isolation, reduction, and acid hydrolysis, gave ¹⁴C-glucitol in the pulsed dextran, which was significantly decreased in the chased dextran, showing that the d-glucose moieties of sucrose are added to the reducing-ends of the covalently linked growing dextran chains. The molecular size of dextran is shown to be inversely proportional to the concentration of the enzyme, indicating a highly processive mechanism in which d-glucose is rapidly added to the reducing-ends of the growing chains, which are extruded from the active site of dextransucrase. It is also shown how the three conserved amino acids (Asp551, Glu589, and Asp 622) at the active sites of glucansucrases participate in the polymerization of dextran and related glucans from a single active site by the addition of the d-glucose moiety of sucrose to the reducing-ends of the covalently linked glucan chains in a two catalytic-site, insertion mechanism.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Active calcium transport in red cell ghosts resealed in dextran solutions

1.Human erythrocytes when lysed and resealed to Ca in the presence of dextran can be readily separated from the suspending medium by low-speed centrifugation. 2. Ghosts trapped Ca and EGTA at the same ratio as present in the haemolytic medium and remained tight to Ca after washing and subsequent incubation for up to 90 min at 37°C. 3. Ca extrusion could be promoted by substrates other than ATP only from ghosts that had been loaded with low free Ca concentrations (1–22 μM). The order of activation by the various substrates employed was $\text{ATP}\text{>}\text{adenine}\text{+}\text{inosine}\text{>}\text{inosine}$. 4. The kinetics of extrusion depended markedly on internal free Ca. The system showed a high affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{3 μ}\text{M}$; $\text{V = 0.34 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at low concentrations (1–22 μM) and a low affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{250 μ}\text{M}$; $\text{V = 0.17 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at high concentrations (0.2–4.0 mM). 5. Both at low and at high free Ca, La-sensitive ATP hydrolysis was closely correlated with La-dependent Ca efflux, in keeping with an stoichiometry of 1. 6. The rate of extrusion was maximal in the presence of 160 mM KCl and decreased to various extents when K was fully replaced by different cations, following the order $\text{K}\text{>}\text{Na = choline}\text{>}\text{Mg}$. 7. The efflux rate of high-K ghosts, resealed to alkaline cations, was stimulated by external Na, whilst Mg and choline were practically without effect. 8. The results indicate that human red cells possess a powerful Ca extrusion mechanism, the activity of which can be modulated by alkaline cations.     

Estimation of the percolation thresholds in lobenzarit disodium native dextran matrix tablets

The objective of the present work is to estimate for the first time the percolation threshold of a new series of dextran (native dextran of high molecular weight [B110-1-2, Mw = 2 x 10(6)]), in matrices of lobenzarit disodium (LBD) and to apply the obtained result to the design of hydrophilic matrices for the controlled delivery of this drug. The formulations studied were prepared with different amounts of excipient in the range of 20% to 70% wt/wt. Dissolution studies were performed using the paddle method (100 rpm) and one face water uptake measurements were performed using a modified Enslin apparatus. The Higuchi, zero-order, and Hixson-Crowell models as well as the nonlinear regression model were employed as empiric methods to study the release data. Values of diffusion exponent 0.563 < n < 0.786 (Korsmeyer equation) for dissolution profile and water uptake mechanism 0.715 < n < 1 (Davidson and Peppas equation) suggested anomalous or complex mechanisms. On the other hand, the contribution of the relaxation or erosion and of the diffusive mechanism in Peppas-Sahlin equation indicated that the main mechanism for drug delivery from tablets is swelling controlled delivery (K(r)/K(d) < 1). The critical points observed in kinetic parameters above 58.63% vol/vol of native dextran B110-1-2 plus initial porosity in the LBD-dextran matrices with a relative polymer/drug particle size of 4.17 were attributed to the existence of an excipient percolation threshold.