Calcium Alginate Microspheres Research Articles

Phagocytosis‐mediated retroviral transduction: co‐internalization of deactivated retrovirus and calcium‐alginate microspheres by macrophages

Efficiency of retrovirus-mediated gene transfer has been hampered by short retroviral half-life due to shedding of retroviral envelope proteins which is of utmost importance to the interaction between retrovirus and cell. Calcium-alginate microspheres with average size of 780 nm were prepared by reverse emulsification and characterized by scanning electron microscopy and microelectrophoresis. To obtain deactivated retroviruses produced from 293 packaging cells, retrovirus-containing media were pre-incubated at 37 degrees C for 6, 12, 18, and 24 h, respectively. Murine J774A.1 macrophages were co-treated with Ca-alginate microspheres and deactivated retroviruses encoding the enhanced green fluorescent protein (eGFP) gene. Through phagocytosis, deactivated retroviruses and Ca-alginate microspheres were co-internalized into macrophages. After retroviral transduction for 24 and 48 h, the percentages of macrophages with eGFP expression were determined by bright-field and fluorescence microscopy. After 48-h incubation with Ca-alginate microspheres and deactivated retroviruses, phagosomes turned into large vacuoles occupied almost half of the cytoplasmic space. This was probably attributed to the erosion of Ca-alginate microspheres by destructive agents within vacuolar compartments and concomitant osmotic swelling. It was reasoned that deactivated retroviruses escaped such enlarged vesicles easily and underwent reverse transcription in the cytosol. The expression of eGFP in macrophages infected by retroviruses pre-incubated for 24 h in the presence of Ca-alginate microspheres was thereby augmented up to tenfold in comparison with the cells treated with 24-h deactivated retroviruses only. Ca-alginate microspheres performed as auxiliary agents for the enhancement of retrovirus-mediated gene transfer efficiency even though retroviruses had been deactivated due to the loss of envelope proteins.

Encapsulation of glucose oxidase and an oxygen-quenched fluorophore in polyelectrolyte-coated calcium alginate microspheres as optical glucose sensor systems

Microspheres coated with polyelectrolyte multilayers (PEM's) are being investigated for potential use as implantable biosensors-so-called "smart tattoos." In this work, the feasibility of this approach for glucose sensors was demonstrated by glucose oxidase encapsulated within calcium alginate microspheres, followed by entrapment of an oxygen-quenched ruthenium compound in the same microstructure. A novel feature of these microdevices is the formation of multilayer nanofilms on the surface of the microspheres, used to stabilize enzyme entrapment and control substrate diffusion. Confocal microscopy was used to confirm the stable encapsulation of sensor chemistry. The reversible response of sensors to step changes in glucose was observed, and preliminary experimental data were compared to theoretical predictions produced by a computational model. These findings demonstrate the promise of the described nanoengineering approach for production of functional implantable glucose sensor materials.

Synthesis of a Kunitz-type serine proteinase inhibitory protein that shares homology with bovine pancreatic trypsin inhibitor by ovine intervertebral disc cells in serum-free alginate bead culture.

The objective of this study was to determine whether disc cells could be cultured under serum-free conditions and whether they synthesized bovine pancreatic trypsin inhibitor (BPTI)-like serine proteinase inhibitory proteins (SPIs) previously demonstrated for ovine chondrocytes. Intervertebral discs from 1- to 2-year-old merino wether sheep were dissected into the annulus fibrosus and nucleus pulposus, and the cells isolated by sequential enzymatic digestion. The cells were grown encapsulated in calcium alginate microspheres under serum-free conditions for 10 days. They remained more than 92% viable as assessed using the vital fluorescent dyes chloromethyl fluorescein diacetate and ethidium homodimer-1 to delineate live/dead cells, respectively. Western and affinity blotting identified a 12-16-kDa media SPI and an additional 34-36-kDa BPTI-like species in solubilized bead samples. This study has demonstrated ovine disc cells synthesized BPTI-like SPIs in serum-free alginate bead culture similar to chondrocyte SPIs; however, the 58-kDa precursor SPI form was not detected suggesting differences in the endogenous processing of these SPIs.

Effects of aldehydes and methods of cross-linking on properties of calcium alginate microspheres prepared by emulsification

Calcium alginate microspheres were prepared by an emulsification method and cross-linked with various aldehydes using different methods. Methanal and pentanedial produced low aggregation of microspheres while octanal and octadecanal produced the opposite effect. The latter two aldehydes displaced very little calcium ions from the alginate microspheres, indicating that the aggregation was due to the tackiness imparted by the aldehydes to the microsphere surface. Higuchi's model was not applicable to the drug release from microspheres in this study. The microspheres treated with methanal or pentanedial showed comparable dissolution T 75% values which were significantly higher than that of the control. In contrast, octanal and octadecanal produced microspheres with lower dissolution T 75% values. The drug contents of the microspheres treated with aldehydes were significantly lower than that of the control. There was insignificant interaction between the aldehydes and the drug. However, the aldehydes were found to impart acidity to the aqueous solution to varying extents, resulting in varying drug loss from the microspheres. The properties of the microspheres were also markedly affected by the method of incorporating the aldehyde. Soaking the microspheres in methanal solution produced microspheres with marked aggregation and low drug content.

Calcium Alginate Microspheres of Bacillus subtilis

Microspheres of Bacillus subtilis were prepared using sodium alginate. Some typical properties of microencapsulated systems, such as microorganism content, particle size, and germination time, were studied. Calcium alginate microspheres were obtained by the emulsification method, dripping into a solution of calcium salt. The conditions of the preparation steps were very soft to produce calcium alginate microspheres containing cells with no apparent changes in general biological properties. The hydrogel matrix provides protection without preventing communication with the surrounding medium.

Differential Expression of Proteoglycan Epitopes and Growth Characteristics of Intervertebral Disc Cells Grown in Alginate Bead Culture

This study assessed the growth characteristics and proteoglycan (PG) production in vitro of cells isolated from various zones; annulus fibrosus (AF), the transitional zone (TZ, an opalescent region situated between the AF and nucleus pulposus, NP) and the NP of the ovine intervertebral disc. The isolated cells were subsequently cultured in calcium alginate microspheres. Cellular metabolic activity was assessed over the 10 days of culture by the bioreduction of a tetrazolium dye substrate to a coloured formazan chromophore which could be measured colorimetrically at 490 nm. These data were correlated with the DNA content of beads measured using the fluorescent dye Hoechst 33258. Viable and non-viable cells in alginate beads were also assessed histochemically using the fluorescent dyes 5-chloromethylfluorescein diacetate and ethidium homodimer-1. PGs synthesised in culture were examined immunohistologically using monoclonal antibodies to defined glycosaminoglycan side chain PG epitopes. The results obtained clearly showed that AF and TZ cells differed from NP cells in the measured indices of cellular metabolism and in the extent of matrix deposition. In contrast to cells from the NP the cells from the AF and TZ were more metabolically active and programmed more to cellular proliferation than to matrix production.

Differential expression of proteoglycan epitopes by ovine intervertebral disc cells in calcium alginate microspheres

Differential expression of proteoglycan epitopes by ovine intervertebral disc cells in calcium alginate microspheres

Effects of poly(vinylpyrrolidone) and ethylcellulose on alginate microspheres prepared by emulsification

Calcium alginate microspheres were prepared by an emulsification process. The effects of two co-polymers, namely poly(vinylpyrrolidone) and ethylcellulose, on the properties of the microspheres were studied. Microspheres prepared with and without poly(vinylpyrrolidone) were spherical and discrete. The microspheres containing poly(vinylpyrrolidone) exhibited a better flow property but the drug content was lower and the drug release rate higher. The method of incorporating poly(vinylpyrrolidone) was found to (significantly) affect the size distribution and drug content of the microspheres. Ethylcellulose produced marked aggregation of the microspheres which also showed a lower drug content, but a slower drug release. The retardation in drug release was attributed to the formation of aggregated microspheres with a less permeable matrix. The addition of triethyl citrate, which is a water-soluble plasticizer, was found to increase the rate of drug release while the use of a higher viscosity grade of ethylcellulose produced the opposite effect. Ethylcellulose improved the flowability of the microspheres to a greater extent than poly(vinyl-pyrrolidone).

Bioadhesive microspheres: III. An in vivo transit and bioavailability study of drug-loaded alginate and poly(fumaric-co-sebacic anhydride) microspheres

Bioadhesive drug delivery systems (BDDSs) could improve bioavailability by protecting bioactive molecules from physical and chemical degradation, enhancing absorption rates by minimizing diffusion barriers, and increasing the period for absorption by prolonging residence time. The in vivo bioadhesive performance of calcium alginate microspheres and poly(fumaric-co-sebacic anhydride) 20:80 microspheres were evaluated in two ways. Firstly, effect on GI transit was measured in rats. P(FA:SA) 20:80 microspheres showed significantly prolonged retention in the gut when compared to alginate microspheres. Secondly, the ability of these polymers to improve relative bioavailability of a model drug, dicumarol, in rats was assayed. A significant increase was measured in the area under the plasma concentration-time curve for dicumarol encapsulated in P(FA:SA) 20:80 when compared to its controls. The results of this study suggest that polymers which have been shown to produce strong bioadhesive forces in vitro and delayed GI transit in vivo may be used to improve oral bioavailability of certain drugs.

Prolonged retention of cross-linked trypsin in calcium alginate microspheres

Trypsin microencapsulated in a calcium alginate matrix was lost quickly through diffusion when the microspheres were placed in an aqueous medium. This problem was overcome by first reacting trypsin with glutaraldehyde to form cross-linkages and then incorporating the enzyme in the alginate micro-spheres. The performance of the cross-linked trypsin remained optimal at pH 8 while it was found to be more heat-stable and remained highly active even at 80°C. Esters and amides of L-arginine were preferentially hydrolysed by the enzyme indicating that cross-linking did not adversely affect the conformation of the active site. There was a suppression in enzymatic activity when the microspheres were placed in reaction media with an increasing concentration of organic solvent such as ethanol, acetonitrile or isopropanol. However, when returned to a totally aqueous environment, the enzyme resumed its initial tryptic capability. Such a microencapsulated form of cross-linked enzyme may find application in enzyme replacement therapy, optical resolution of racemic compounds as well as organic synthesis in an aqueous-organic environment.

Cellular Response to Transforming Growth Factor-β1 and Basic Fibroblast Growth Factor Depends on Release Kinetics and Extracellular Matrix Interactions

The extracellular matrix plays an important role in growth factor biology, serving as a potential platform for rapid growth factor mobilization or a sink for concentrated sequestration. We now demonstrate that when a growth factor binds reversibly to the matrix, its effects are augmented by this interaction, and when the factor is absorbed irreversibly to the extracellular matrix, it becomes sequestered. These findings call into question the notion that all growth factors are best presented to cells and tissues in a sustained and controlled fashion. In our studies, we examined basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) release kinetics from synthetically fabricated microsphere devices and naturally synthesized extracellular matrix. While the sustained release of bFGF was up to 3.0-fold more potent at increasing vascular endothelial and smooth muscle cell proliferation than bolus administration, the reverse was true for TGF-beta1. A bolus of TGF-beta1 inhibited vascular cells up to 3.8-fold more efficiently than the same amount of TGF-beta1 if control-released. Both growth factors bound to the extracellular matrix, but only bFGF was released in a controlled fashion (2.8%/day). Contact with the extracellular matrix and subsequent release enhanced bFGF activity such that it was 86% more effective at increasing smooth muscle cell numbers than equal amounts of growth factor diluted from frozen stock. TGF-beta1 remained tightly adherent. The small amount of TGF-beta1 released from the extracellular matrix was approximately 30% less effective than bolus administration at inhibiting vascular endothelial and smooth muscle cell growth. Sustained growth factor release may be the preferable mode of administration, but only when a similar mode of metabolism is utilized endogenously.

Surfactant effects on alginate microspheres

In the production of calcium alginate microspheres involving an emulsification process, the surfactants incorporated can affect the formation of the microspheres. In this study, the influence of the blend of surfactants, hydrophile-lipophile balance (HLB) and the sequence of addition of surfactant on the properties of the microspheres was determined. The findings showed that maintaining the same HLB, surfactants with more fatty acid chains produced larger microspheres which also have higher drug content, whereas for surfactants with longer polyoxyethylene chains, the microspheres were smaller. Varying the HLB, whilst keeping the same blend of surfactants, resulted in changes in the size and shape of the microspheres. The incorporation of the hydrophilic surfactant into the aqueous phase containing the drug gave rise to larger microspheres. These had a lower drug encapsulation efficiency. The drug release rate was faster, and this is attributed to the presence of unencapsulated drug crystals in the product.

Communications Drug Carriers for Transdermal Preparations of Flurbiprofen

Flurbiprofen was incorporated in different carriers like sodium alginate gel, calcium alginate microspheres, gelatin nanoparticles and complex with betacyclodextrin and incorporated in polyethylene glycol bases. Pharmacodynamic and bioavailability studies were carried out in male rats. It was found that drug incorporated in sodium alginate gel and drug complexed with betacyclodextrin were found to be suitable for designing tranadermal preparations since they resulted in better therapeutic efficacy.

Influence of hydrophile-lipophile balance on alginate microspheres

In this study, calcium alginate microspheres were prepared by an emulsification method using surfactants. A blend of sorbitan trioleate and polyoxyethylene sorbitan trioleate of varying HLB was used to produce the microspheres. The results obtained showed that the surfactant concentration and HLB played an important role in the formation of the microspheres. The size, size distribution and to a certain extent the shape were significantly affected by the HLB. However, HLB did not alter the drug encapsulation efficiency appreciably but did affect the rate of drug release.

Drug encapsulation in alginate microspheres by emulsification.

A method based on an emulsification process was developed for the production of calcium alginate microspheres. Isopropyl alcohol and acetone, which are strong dehydrating agents, were used to aid in the hardening and drying of the microspheres. However, the amount of drug encapsulated was very low. This was due to the drug being soluble in the dehydrating solvents. In the absence of the solvents a high percentage of drug was encapsulated, and this was further increased by forming the microspheres by phase inversion. It was also found that a suspension of the drug particles was required for effective microencapsulation. The efficiency of drug encapsulation generally increased with the ratio of drug to encapsulating material. The microspheres produced were free-flowing and most of them were smaller than 150 microns.