Enhancement of the Stability of Insoluble Calcium Particles Using a Phospholipid Coating

Calcium is one of the most important elements in the human body. Insoluble calcium particles are often used in calcium-fortified food products, such as calcium-fortified milk, dairy beverages or protein powders. However, their suspension may be unstable often leading to precipitation in such products. In this study, three different kinds of insoluble calcium particles, i.e. hydroxyapatite (HA), tricalcium phosphate (TCP) and calcium carbonate (CaCO₃) were coated with amphiphilic phospholipids using a solvent-exchange method. Suspension stability of these insoluble calcium particles was effectively improved with phospholipid coating, especially for HA and TCP, as more phospholipids were coated on the surface of these two calcium particles than CaCO₃. Phospholipid coating increased the electrostatic repulsions between particles, preventing the particles from aggregating and precipitating. In addition, the digestibility of phospholipid-coated insoluble calcium particles was tested in simulated gastric juice, and the dissolution time of these insoluble calcium particles was prolonged through phospholipid coating.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Critical Point Drying Method Using Dry Ice

A critical point drying method using dry ice (solid carbon dioxide) instead of liquid carbon dioxide is reported. After the specimens are placed in the chamber of the medical point drying apparatus, dry ice cut to the shape of the chamber is inserted. The chamber is closed and warmed to change the dry ice into liquid carbon dioxide. This method needs no gas cylinder and even minute or fragile specimens do not blow away because there is no flow of gas. This method can be used with any kind of critical point drying apparatus.     

A Statistical Approach to Optimize the Spray Drying of Starch Particles: Application to Dry Powder Coating

This article describes the preparation of starch particles, by spray drying, for possible application to a dry powder coating process. Dry powder coating consists of spraying a fine powder and a plasticizer on particles. The efficiency of the coating is linked to the powder morphological and dimensional characteristics. Different experimental parameters of the spray-drying process were analyzed, including type of solvent, starch concentration, rate of polymer feeding, pressure of the atomizing air, drying air flow, and temperature of drying air. An optimization and screening of the experimental parameters by a design of the experiment (DOE) approach have been done. Finally, the produced spray-dried starch particles were conveniently tested in a dry coating process, in comparison to the commercial initial starch. The obtained results, in terms of coating efficiency, demonstrated that the spray-dried particles led to a sharp increase of coating efficiency value.     

Preparation and In Vitro Aerosol Performance of Spray-Dried Shuang-Huang-Lian Corrugated Particles in Carrier-Based Dry Powder Inhalers

The development of dry powder inhalation (DPI) products of traditional Chinese medicine (TCM) remains to be a challenge due to chemical complexity and batch-to-batch variations in constituent composition. This study was to investigate the feasibility of using spray-dried corrugated particles to improve the aerodynamic performance of a TCM, Shuang-Huang-Lian (SHL), in carrier-based DPI. Particles with different surface roughness were spray-dried by the addition of leucine and concomitant manipulation of spray-drying parameters. The surface roughness was determined by atomic force microscopy, whilst the aerodynamic performance of drug particle–mannitol/lactose blends was evaluated using a next-generation pharmaceutical impactor through a Cyclohaler. Although the emission efficiency for corrugated particle-based DPI was ∼10% lower than that for smooth SHL, the fine particle fractions (FPF_<4.4 μm) of 32.4–36.8% for the former were significantly higher than those of 14.7–16.2% for the latter. In particular, the FPF and fraction of drug detached from the carrier appeared not to be significantly affected by the variation in constituent composition of SHL. This study demonstrates that the use of corrugated particles in carrier-based DPI improved aerosol performance by facilitating drug detachment from the carrier, independent of variation in constituent composition, and such particles were potentially applicable to the development of SHL DPI products.KEY WORDS: dry powder inhaler, Shuang-Huang-Lian, spray-drying, surface roughness, traditional Chinese medicine     

Formulation of Controlled-Release Capsules of Biopharmaceutical Classification System I Drugs Using Niacin as a Model

Vitamin B₃ is made up of niacin (nicotinic acid) and its amide, niacinamide. Both have equivalent vitamin activity, but only niacin (not niacinamide) is effective in lowering elevated low-density lipoprotein cholesterol and triglyceride levels in the blood. Administration of an extended-release (ER) oral tablet would frequently encounter food. If hydrogel is used to formulate the matrix of a biopharmaceutical classification system I drug (high solubility and high permeability), the dosage form absorbs water and swells.. The softened outer layer may be slashed off by food present in the stomach, thus, exposing the core tablet more readily for water absorption and speeding up drug release from its original designed rate. This project aimed to formulate niacin CR pellets made of hydrophobic inert matrix. After niacin was melted with excipients and cooled, the mass was extruded and spheronized into pellets. Size distribution and flowability were determined before pellets were filled into hard gelatin capsule. The USP dissolution study revealed that a candidate formulation of 250 mg in strength released similar amount of niacin as its commercial reference, niacin controlled-release 500 mg tablet, in 6 h (223.9 ± 23.8 mg, n = 4 versus 259.4 ± 2.6 mg, n = 3). The differential scanning calorimetry study of the pellets in capsules stored in 40°C for 4 weeks, and the content assay of capsules in 40°C up to 6 months suggested that niacin was stable within the innovative formulation. In vitro release from this innovative ER capsules stored at 40°C up to 4 weeks were also investigated.     

An Investigation into the Effect of Fine Lactose Particles on the Fluidization Behaviour and Aerosolization Performance of Carrier-Based Dry Powder Inhaler Formulations

The effect of milled and micronized lactose fines on the fluidization and in vitro aerosolization properties of dry powder inhaler (DPI) formulations was investigated, and the suitability of static and dynamic methods for characterizing general powder flow properties of these blends was assessed. Lactose carrier pre-blends were prepared by adding different lactose fines (Lactohale® (LH) 300, 230 and 210) with coarse carrier lactose (Lactohale100) at 2.5, 5, 10 and 20 wt% concentrations. Powder flow properties of lactose pre-blends were characterized using the Freeman Technology FT4 and Schulze RST-XS ring shear tester. A strong correlation was found between the basic flow energy (BFE_Norm) measured using the Freeman FT4 Rheometer and the flowability number (ff_c) measured on Schulze RST-XS. These data indicate that both static and dynamic methods are suitable for characterizing general powder flow properties of lactose carriers. Increasing concentration of fines corresponded with an increase in the normalized fluidization energy (FE_Norm). The inclusion of fine particles of lactose resulted in a significant (p < 0.05) increase in fine particle delivery of budesonide and correlated with FE_Norm. This trend was strongest for lactose containing up to 10 wt% LH300. A similar trend was found for the milled lactose grades LH230 and LH210. However, the increase in FE_Norm upon addition of milled fines only corresponded to a very slight improvement in the performance. These data suggest that whilst the fluidization energy correlated with fine particle delivery, this relationship is specific to lactose grades of similar particle size.KEY WORDS: dry powder inhaler, fluidisation, lactose, powder flow     

Preparation of Mesoporous Nano-Hydroxyapatite Using a Surfactant Template Method for Protein Delivery

Mesoporous nano-hydroxyapatite (n-HA) has gained more and more attention as drug storage and release hosts.The aim of this study is to observe the effect of the ratio of surfactant to the theoretical yield of HA on the mesoporous n-HA,then to reveal the effect of the mesoporous nanostrueture on protein delivery.The mesoporous n-HA was synthesized using the wet precipitation in the presence of cetyltrimethylammonium bromide (CTAB) at ambient temperature and normal atmospheric pressure.The morphology,size,crystalline phase,chemical composition and textural characteristics of the product were well characterized by X-ray Powder Diffraction (XRD),Fourier Transform Infrared Spectroscopy (FTIR),Scanning Electron Microscopy (SEM),Transmission Electron Microscopy (TEM),Dynamic Light Scattering (DLS) and N2 adsorption/desorption,respectively.The protein adsorption/release studies were also carried out by using Bovine Serum Albumin (BSA) as a model protein.The results reveal that the mesoporous n-HA synthesized with CTAB exhibits high pure phase,low crystallinity and the typical characteristics of the mesostructure.The BSA loading increases with the specific surface area and the pore volume of n-HA,and the release rates of BSA are different due to their different pore sizes and pore structures,n-HA synthesized with 0.5％ CTAB has the highest BSA loading and the slowest release rate because of its highest surface area and smaller pore size.These mesoporous n-HA materials demonstrate a potential application in the field of protein delivery due to their bioaetive,biocompatible and mesoporous properties.     

A New Role of Fine Excipient Materials in Carrier-Based Dry Powder Inhalation Mixtures: Effect on Deagglomeration of Drug Particles During Mixing Revealed

The potential of fine excipient materials to improve the performance of carrier-based dry powder inhalation mixtures is well acknowledged. The mechanisms underlying this potential are, however, open to question till date. Elaborate understanding of these mechanisms is a requisite for rational rather than empirical development of ternary dry powder inhalation mixtures. While effects of fine excipient materials on drug adhesion to and detachment from surfaces of carrier particle have been extensively investigated, effects on other processes, such as carrier–drug mixing, capsule/blister/device filling, or aerosolization in inhaler devices, have received little attention. We investigated the influence of fine excipient materials on the outcome of the carrier–drug mixing process. We studied the dispersibility of micronized fluticasone propionate particles after mixing with α-lactose monohydrate blends comprising different fine particle concentrations. Increasing the fine (D < 10.0 μm) excipient fraction from 1.84 to 8.70% v/v increased the respirable drug fraction in the excipient–drug mixture from 56.42 to 67.80% v/v (p < 0.05). The results suggest that low concentrations of fine excipient particles bind to active sites on and fill deep crevices in coarse carrier particles. As the concentration of fine excipient particles increases beyond that saturating active sites, they fill the spaces between and adhere to the surfaces of coarse carrier particles, creating projections and micropores. They thereby promote deagglomeration of drug particles during carrier–drug mixing. The findings pave the way for a comprehensive understanding of contributions of fine excipient materials to the performance of carrier-based dry powder inhalation mixtures.     

Fine Mapping Causal Variants with an Approximate Bayesian Method Using Marginal Test Statistics

Two recently developed fine-mapping methods, CAVIAR and PAINTOR, demonstrate better performance over other fine-mapping methods. They also have the advantage of using only the marginal test statistics and the correlation among SNPs. Both methods leverage the fact that the marginal test statistics asymptotically follow a multivariate normal distribution and are likelihood based. However, their relationship with Bayesian fine mapping, such as BIMBAM, is not clear. In this study, we first show that CAVIAR and BIMBAM are actually approximately equivalent to each other. This leads to a fine-mapping method using marginal test statistics in the Bayesian framework, which we call CAVIAR Bayes factor (CAVIARBF). Another advantage of the Bayesian framework is that it can answer both association and fine-mapping questions. We also used simulations to compare CAVIARBF with other methods under different numbers of causal variants. The results showed that both CAVIARBF and BIMBAM have better performance than PAINTOR and other methods. Compared to BIMBAM, CAVIARBF has the advantage of using only marginal test statistics and takes about one-quarter to one-fifth of the running time. We applied different methods on two independent cohorts of the same phenotype. Results showed that CAVIARBF, BIMBAM, and PAINTOR selected the same top 3 SNPs; however, CAVIARBF and BIMBAM had better consistency in selecting the top 10 ranked SNPs between the two cohorts. Software is available at https://bitbucket.org/Wenan/caviarbf.     

A Simple Method for Preparation of Poly-mannuronate Using Poly-guluronate Lyase

A simple method for preparation of poly-mannuronate from alginate has been developed. By making the best use of the substrate specificity of a poly-guluronate lyase, we prepared a poly-mannuronate of which the properties were almost identical to those of the poly-mannuronate produced by Haug’s acid hydrolysis method. Our method is very useful in terms of time and labor saving.     

Effect of Surface Coating with Magnesium Stearate via Mechanical Dry Powder Coating Approach on the Aerosol Performance of Micronized Drug Powders from Dry Powder Inhalers

The objective of this study was to investigate the effect of particle surface coating with magnesium stearate on the aerosolization of dry powder inhaler formulations. Micronized salbutamol sulphate as a model drug was dry coated with magnesium stearate using a mechanofusion technique. The coating quality was characterized by X-ray photoelectron spectroscopy. Powder bulk and flow properties were assessed by bulk densities and shear cell measurements. The aerosol performance was studied by laser diffraction and supported by a twin-stage impinger. High degrees of coating coverage were achieved after mechanofusion, as measured by X-ray photoelectron spectroscopy. Concomitant significant increases occurred in powder bulk densities and in aerosol performance after coating. The apparent optimum performance corresponded with using 2% w/w magnesium stearate. In contrast, traditional blending resulted in no significant changes in either bulk or aerosolization behaviour compared to the untreated sample. It is believed that conventional low-shear blending provides insufficient energy levels to expose host micronized particle surfaces from agglomerates and to distribute guest coating material effectively for coating. A simple ultra-high-shear mechanical dry powder coating step was shown as highly effective in producing ultra-thin coatings on micronized powders and to substantially improve the powder aerosolization efficiency.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

Torsion Profiling of Proteins Using Magnetic Particles

We report a method to profile the torsional spring properties of proteins as a function of the angle of rotation. The torque is applied by superparamagnetic particles and has been calibrated while taking account of the magnetization dynamics of the particles. We record and compare the torsional profiles of single Protein G-Immunoglobulin G (IgG) and IgG-IgG complexes, sandwiched between a substrate and a superparamagnetic particle, for torques in the range between 0.5 × 10³ and 5 × 10³ pN·nm. Both molecular systems show torsional stiffening for increasing rotation angle, but the elastic and inelastic torsion stiffnesses are remarkably different. We interpret the results in terms of the structural properties of the molecules. The torsion profiling technique opens new dimensions for research on biomolecular characterization and for research on bio-nanomechanical structure-function relationships.     

Fine Needle Aspiration of Palpable Breast Lumps: A 1-Year Audit Using the Cytospin Method

A fine-needle aspiration (FNA) service for the diagnosis of palpable breast lumps was started at the Royal Preston Hospital, Preston, UK, in November 1989. Over the subsequent year, 407 FNAs were taken from 393 women. A simple technique was used which involved the surgeon flushing the aspirate into 10 ml of Cytospin collection fluid; cytocentrifuge preparations were then safely and conveniently prepared in the laboratory. Slides were stained with Papanicolaou and H&E. The method detected 112 out of a total of 121 cancers (92.6%); of the nine that were undetected, five aspirates were inadequate and four were falsely reported as negative. There were no false positives. The overall inadequate rate was 11.0%. Excluding inadequate samples, the absolute sensitivity was 89.7% and complete sensitivity 96.6% with 94.4% specificity. This 1-year audit has shown the Cytospin method of FNA in palpable breast disease to have a favourable sensitivity and specificity, and therefore to be an alternative to conventional FNA using direct smears.     

A Feasibility Study on Pellet Coating Using a High-Speed Quasi-continuous Coater

Pellet coating is traditionally carried out using the Wurster coater. This study investigated the feasibility of pellet coating in a newly developed coater built with a unique airflow system, the Supercell™ coater (GEA Pharma Systems, UK). A full factorial design study was carried out to evaluate the influences of the spray rate of the coating dispersion, batch size of the pellet load, pellet size fraction and plenum pressure of the fluidizing air on the color coating of pellets in the Supercell™ coater. Results showed that pellets could be successfully coated using the Supercell™ coater. Higher plenum pressures and lower spray rates were found to minimize pellet agglomeration during coating. Although coating efficiencies were comparable amongst the different pellet size fractions, larger batch sizes of pellets were coated with higher efficiencies. Process optimization was carried out for each pellet size fraction and a large batch size (120 g) in combination with a high plenum pressure (1,500 mm WC) were deemed optimal. Optimal spray rates differed according to pellet size fraction and a lower spray rate was required for smaller pellets. Pellet flow patterns observed during coating were dependent on the pressure drop across the fluidized load. A ‘swirling’ pellet flow pattern was generally observed at coating conditions which led to optimal outcomes.KEY WORDS: fluid bed, fluidization, pellet coating, pellet flow patterns, pressure drop, process optimization, Supercell™ coater     

以重组人巨细胞病毒pp65制备的多克隆抗体建立检测感染性病毒颗粒方法

人巨细胞病毒(HCMV)活动性感染是造成器官移植失败的重要原因。建立灵敏、特异的检测方法可为及早发现感染、及时给予抗病毒治疗提供依据。以离子交换和分子筛方法纯化重组表达的HCMVpp65(rp65hp)抗原,经两步纯化后,rp65hp纯度达到95%。免疫家兔制备的抗血清,ELISA抗体滴度高达1∶2560000;免疫印迹证明能与HCMVpp65抗原特异反应。将HCMV病毒感染细胞,以纯化的多克隆抗体建立了间接免疫荧光法,成功地检测到细胞中的病毒。因此,该方法为临床检测HCMV病毒活动性感染奠定了良好的基础。     

Fine Mapping in 94 Inbred Mouse Strains Using a High-Density Haplotype Resource

The genetics of phenotypic variation in inbred mice has for nearly a century provided a primary weapon in the medical research arsenal. A catalog of the genetic variation among inbred mouse strains, however, is required to enable powerful positional cloning and association techniques. A recent whole-genome resequencing study of 15 inbred mouse strains captured a significant fraction of the genetic variation among a limited number of strains, yet the common use of hundreds of inbred strains in medical research motivates the need for a high-density variation map of a larger set of strains. Here we report a dense set of genotypes from 94 inbred mouse strains containing 10.77 million genotypes over 121,433 single nucleotide polymorphisms (SNPs), dispersed at 20-kb intervals on average across the genome, with an average concordance of 99.94% with previous SNP sets. Through pairwise comparisons of the strains, we identified an average of 4.70 distinct segments over 73 classical inbred strains in each region of the genome, suggesting limited genetic diversity between the strains. Combining these data with genotypes of 7570 gap-filling SNPs, we further imputed the untyped or missing genotypes of 94 strains over 8.27 million Perlegen SNPs. The imputation accuracy among classical inbred strains is estimated at 99.7% for the genotypes imputed with high confidence. We demonstrated the utility of these data in high-resolution linkage mapping through power simulations and statistical power analysis and provide guidelines for developing such studies. We also provide a resource of in silico association mapping between the complex traits deposited in the Mouse Phenome Database with our genotypes. We expect that these resources will facilitate effective designs of both human and mouse studies for dissecting the genetic basis of complex traits.PHENOTYPIC variation among inbred mouse strains exposed to a disease-causing agent (be it genetic, infectious, or environmental) provides potential insight into human disease processes that often cannot be practically achieved through direct human studies. Indeed, hundreds of phenotype measurements related to human diseases are available for dozens of inbred strains in common use over the past 50–100 years (Bogue et al. 2007; Grubb et al. 2009). As with the direct study of chronic disease in humans, key steps toward determining the genetic underpinnings of this phenotypic variation are to develop a catalog of the genetic variation among inbred mouse strains and to interpret the structure of variation patterns across the strains. Recent advances in high-throughput genotyping and DNA resequencing technologies are making it possible to rapidly uncover the genetic variation maps of many model organisms (Lindblad-Toh et al. 2005; Mackay and Anholt 2006; Borevitz et al. 2007; Frazer et al. 2007; International Hapmap Consortium 2007; Star Consortium 2008). A recent whole-genome resequencing study of 15 inbred mouse strains captured a significant fraction of the genetic variation among a limited number of strains, allowing researchers to infer patterns of genetic variation and to identify the ancestral origin of the genetic variation (Frazer et al. 2007; Yang et al. 2007). Yet the availability and common experimental employment of hundreds of inbred strains, including >190 stocks available from the Jackson Laboratory, motivates the need for a high-density variation map for a larger set of strains. We have assembled the Mouse HapMap, a resource consisting of a dense set of genotypes for a total of 138,980 unique biallelic single nucleotide polymorphisms (SNPs) in 94 inbred mouse strains at an average spacing of 20 kb on chromosomes 1–19 and X.This resource is ideal for performing high-resolution mapping studies under QTL peaks. We evaluate the feasibility and effectiveness of such studies by examining a typical study from the Mouse Phenome Database (MPD) (Bogue et al. 2007; Grubb et al. 2009) (http://www.jax.org/phenome) and measure the statistical power to detect genetic associations in regions of various sizes. We provide several resources to the mouse genetics community for supporting such studies and a webserver that can estimate the significance threshold, compute the statistical power of a proposed study, and perform in the fine mapping of measured phenotypes. In addition, we provide a database of associations for all phenotypes contained in the MPD. The web resources are available at http://mouse.cs.ucla.edu/.