Chitosan nanoparticle-loaded mannitol microspheres: structure and surface characterization

In this work, we aimed to characterize the surface and the internal structure of mannitol microspheres containing chitosan/tripolyphosphate nanoparticles, which were prepared by spray-drying. These microspheres were recently proposed as valuable candidates to transport therapeutic protein-loaded nanoparticles to the lungs owing to their favorable aerodynamic properties. To observe the distribution of chitosan nanoparticles and mannitol in the microspheres, specific characterization techniques, such as confocal laser scanning microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry, were used. Results showed that mannitol is distributed in the whole particle and nanoparticles are homogeneously mixed with mannitol. Moreover, both components were detected in the microsphere surface, mannitol being present to a higher extent, which is in agreement with the theoretical mannitol/nanoparticle ratio of microspheres (80/20). Therefore, this work confirmed that chitosan nanoparticles were successfully encapsulated in mannitol microspheres, providing a homogeneous distribution of the nanoparticles and, hence, of the nanoencapsulated therapeutic macromolecule.     

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.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

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.     

Synthesis and characterization of cross-linked malonylchitosan microspheres for controlled release of acyclovir

The purpose of the present study was to obtain a polymeric system for delayed release of the drug acyclovir (ACV), which can be used for treatment of Herpes simplex and Varicella Zoster. The gelled chitosan (GCT) microspheres were obtained by coacervation-phase separation. They were treated with malonic acid to obtain malonylchitosan (MLCT) microspheres, which were characterized by, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (¹³C NMR), elemental analysis (CHN), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The drug was encapsulated in MLCT microspheres by a contact adsorption technique, and the final formulation (MLCT-ACV), was analyzed for loading efficiency, degree of swelling and in vitro release profiles. The results obtained support the N-substitution of malonyl groups in the MLCT microspheres. The loading efficiency increased with impregnation time and a major amount of drug was encapsulated after 24 h. The swelling rate was higher in acid pH. The median release time was 5.5 h in pH 1.2 and 6.8. The mechanism involved in release was non-Fickian (0.43 < n < 0.85, n = 0.8474) and Super Case II kinetics (n > 1, n = 1.0491) at pH 1.2 and 6.8, respectively.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

New intrinsically radiopaque hydrophilic microspheres for embolization: synthesis and characterization

Polymeric particles currently used for embolization procedures have the disadvantage that they are radiolucent, that is, invisible on X-ray images, and consequently the interventional radiologist has to resort to angiography to (indirectly) monitor the fate of the particles. Here, we introduce intrinsically radiopaque hydrophilic microspheres. Since these microspheres can directly be visualized on X-ray images, using these microspheres for embolization purposes will allow superprecise location of the embolic material, both during and after the procedure. The microspheres, which are prepared by suspension polymerization, are based on the radiopaque monomer 2-[4-iodobenzoyl]-oxo-ethylmethacrylate and hydroxyethylmethacrylate (HEMA) and/or 1-vinyl-2-pyrrolidinone (NVP) as hydrophilic component. It has been shown that for clinically relevant X-ray visibility the spheres should contain at least 20 wt % iodine. At this iodine content, copolymerization with HEMA results in spheres that hardly imbibe water (EQ = 1.08). When HEMA is replaced by NVP, the volume swelling ratio can be significantly increased (to 1.33).     

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.     

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.     

Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy

Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.     

Preparation and characterization of glutaraldehyde cross-linked O-carboxymethylchitosan microspheres for controlled delivery of pazufloxacin mesilate

O-carboxymethylchitosan (OCMC) microspheres containing an antibiotic drug pazufloxacin mesilate (PM) have been successfully prepared by emulsion cross-linking using glutaraldehyde (GA). Various manufacturing parameters, including amount of cross-linking agent and OCMC:PM ratios were altered to optimize process variables during the microspheres production. The structure and morphology were characterized by Fourier transform infrared (FT-IR), wide-angle X-ray diffraction (WXRD) and scanning electron microscopy (SEM). The swelling and releasing behaviors of the microspheres at pH 1.2 and 7.4 media were investigated. The results revealed that the microspheres had a spherical, rough morphology and with a narrow size distribution. The degree of swelling of microspheres at pH 7.4 media was higher than that at pH 1.2 media. The microspheres proved to be successful in prolonging drug release. The release of PM was found to depend upon the extent of matrix cross-linking and drug loading. The release profiles of PM from OCMC microspheres were found to be biphasic with a burst release followed by a gradual release phase, and followed the Higuchi matrix model.     

Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b

Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.     

Preparation and characterization of native poly(3-hydroxybutyrate) microspheres from Ralstonia eutropha

An efficient process for the preparation of poly(3-hydroxybutyrate) (PHB) microspheres with a narrow size distribution was developed. PHB was produced by a fed-batch culture of Ralstonia eutropha using fructose syrup as the sole carbon source. After autoclaving the bacteria, PHB granules, which accumulated in the cells, were isolated by a detergent/hypochlorite treatment and then spray-dried to obtain the microspheres. The diameters of the PHB microspheres ranged from 0.6 to 1.1 m and the weight-average molecular weights were approximately 50000 with polydispersity indexes of 5.0. The microspheres had a porous internal structure with an average porosity value of 72% and efficiently blocked UV light shorter than 220 nm. When isosorbide dinitrate was used as a model drug, the optimal drug loading concentration of the microspheres for controllable retardation was 3% (w/w). Almost 80% of the loaded drug (3%, w/w) was released within 12 h with typical sustained drug release behaviors.     

Synthesis and characterization of chitosan-based pH-sensitive semi-interpenetrating network microspheres for controlled release of diclofenac sodium

pH-Sensitive semi-interpenetrating networks (IPNs) based on chitosan (Cs) and acrylamide-grafted hydroxyethylcellulose (AAm-g-HEC) were prepared in the form of microspheres (MPs) by emulsion-crosslinking technique using glutaraldehyde (GA) as a crosslinker. Diclofenac sodium (DS) drug was successfully encapsulated into IPN microspheres by varying the ratio of Cs and AAm-g-HEC, % drug loading, and amount of GA. DS encapsulation of up to 83% was obtained as measured by UV spectroscopy. MPs with average particle sizes in the range of 188-310 μm were obtained. MPs were characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), and Differential scanning calorimetry (DSC). Diffusion coefficients (D) of water transport through the microspheres were determined using an empirical equation. In vitro release of DS from these matrices has been investigated in pH 1.2 and 7.4 media.     

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.     

Development and characterization of cholinephosphotransferase antibody

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.     

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.     

Synthesis and characterization of multifunctional poly(glycidyl methacrylate) microspheres and their use in cell separation

Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent, and cancer cell-specific moieties were prepared in four steps: (i) preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylenediamine to obtain amino groups, (ii) precipitation of iron ions (Fe²⁺ and Fe³⁺) to form Fe₃O₄ nanoparticles within the microspheres, (iii) consecutive reactions of folic acid with the amino groups on PGMA, and (iv) incorporation of fluorescein isothiocyanate into the microspheres. The microspheres were superparamagnetic, highly monodispersive, intensively fluorescent, and capable of recognizing and binding cancer cells that overexpress folic acid receptors. It was demonstrated that with these microspheres, HeLa cells could be captured from their suspension and easily moved in the direction of the externally applied magnetic field.     

Curdlan microspheres. Synthesis,characterization and interaction with proteins (enzymes,vaccines)

Microparticles of curdlan, synthesized through crosslinking with epichlorohydrin in organic suspension media, were chemically modified with the aim of introducing strongly and/or weakly acidic anionic and palmitoyl hydrophobic groups. Microparticles of both curdlan and curdlan derivatives were physico-chemically characterized. Study of the interaction with enzymes, such as lysozyme, and vaccines, such as tetanus anatoxin, showed a co-operative protein retention effect, induced by electrostatic and hydrophobic forces. The results of the in vitro release studies on support–protein complexes recommend them as potential controlled release systems.