Large Initial Burst Research Articles

Preparation and In Vitro Evaluation of Chitosan Microspheres Containing Prednisolone: Comparison of Simple and Conjugate Microspheres

The purpose of the present study was to obtain a novel microparticulate formulation of prednisolone, which was adequate for the treatment of inflammatory bowel disease (IBD). The formulations prepared were evaluated in vitro. Two types of chitosan microspheres containing prednisolone, named Ch-Pred and Ch-SP-MS, were prepared by an emulsification-solvent evaporation method using a chitosan-prednisolone mixture and a chitosan-succinyl-prednisolone conjugate (Ch-SP), respectively. Ch-Pred and Ch-SP-MS were obtained in almost spherical shape. Ch-Pred showed a relatively high drug content of 13.2% (w/w), but the particle size was distributed from 10 to 45 µm, and a large initial burst release of approximately 60% was observed. On the other hand, although Ch-SP-MS exhibited a fairly low drug content of 3.5% (w/w), their particle size ranged from several hundred nanometers to 20 µm, with the mean diameter of 5 µm, and a gradual drug release profile was achieved. These characteristics on particle size and in vitro release suggested that Ch-SP-MS should have good potential as a microparticulate system for the treatment of IBD.

Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery

The fabrication, morphological characterization, and drug release kinetics from microspheres of three bioerodible polyanhydrides, poly[1,6-bis( p-carboxyphenoxy)hexane] (poly(CPH)), poly(sebacic anhydride) (poly(SA)), and the copolymer poly(CPH-co-SA) 50:50 (CPH:SA 50:50) is reported. The fabrication technique yields microspheres with different morphologies for each of the three polymers studied, ranging from very smooth exterior surfaces for poly(CPH) to coarse surface roughness with large pores for poly(SA). Release profiles for the model drug, p-nitroaniline are also different for each polymer. The release profile from poly(CPH) has a large initial burst and shows little additional release after 2 days. The release from poly(SA) is nearly zero-order and lasts for about 8 days. The release profile from CPH:SA 50:50 shows a relatively small burst and then exhibits zero-order release for about 1 month. The different release profiles are attributed to both polymer erosion rates and drug distribution characteristics of the microspheres. Tailored release profiles of a burst followed by zero-order release are obtained by appropriately combining the microspheres. This technique enables independent modulation of both the burst and the zero-order release rate by varying the number of poly(CPH) and poly(SA) microspheres respectively. Additionally, the zero-order release can be extended from about a week to a month by including CPH:SA 50:50 microspheres.

Fabrication, characterization and in vitro release of paclitaxel (Taxol ®) loaded poly (lactic-co-glycolic acid) microspheres prepared by spray drying technique with lipid/cholesterol emulsifiers

Spray dry technique was applied to produce paclitaxel loaded microspheres of biodegradable poly (lactic-co-glycolic acid) (PLGA) as an alternative delivery system. Various emulsifiers such as l-α-dipalmitoyl-phosphatidylcholine (DPPC), cholesterol, polyvinyl alcohol (PVA), gelatin were incorporated in order to achieve high encapsulating efficiency of paclitaxel in the microspheres and desired properties for a sustained release. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the surface of the microspheres with high ratio of lipid was spherical and smooth. Those made with other emulsifiers had rougher surface with pores. Incorporation of lipid, cholesterol or gelatin can significantly increase the drug content in the microspheres. The differential scanning calorimetry (DSC) result indicated that the paclitaxel trapped in the microspheres existed in an amorphous or disordered-crystalline status in the polymer matrix. The zeta potential of the microspheres was negative in general and was strongly influenced by the type of the emulsifiers used in fabrication. The system formulated with cholesterol was most stable. The release profiles of various formulations with PVA, gelatin as well as low ratio of DPPC showed almost zero-order release kinetics in the first 3 weeks after an initial burst less than 5% in the first day. The release rate then gradually decreased. The microspheres fabricated with high ratio of DPPC exhibited large initial burst. When cholesterol was combined together with DPPC as an emulsifier, the release became faster.

Active growth factor delivery from poly( d, l-lactide-co-glycolide) foams prepared in supercritical CO 2

A method for the production of microporous poly( d, l-lactide-co-glycolide) foams containing encapsulated proteins using supercritical carbon dioxide is described. Foams generated as aqueous protein emulsions in a polymer–solvent solution were saturated with carbon dioxide at supercritical conditions, and then suddenly supersaturated at ambient conditions causing bubble nucleation and precipitation of the polymer. Proteins contained in the water phase of the emulsion were encapsulated within the foams, including basic fibroblast growth factor (bFGF), an angiogenic factor of interest in tissue engineering applications. The release and activity of bFGF from these foams was determined in vitro and compared with similar porous scaffolds prepared by traditional solvent casting–salt leaching techniques. Total protein release rate was greater from structures made in CO 2 than those made by the salt leaching technique, however a large initial burst of bFGF was released from the salt leached structures. This initial burst was not observed from the polymer foams processed in CO 2 and active bFGF was released at a relatively constant rate. Residual methylene chloride levels were measured in the foams made with CO 2 and were found to be above the limits imposed by the US Pharmacopoeia implying that further solvent removal would be required prior to in vivo use.

Controlled Release of Bone Growth Factors from Injectable, Biodegradable Polymer Scaffolds for Bone Tissue Engineering

AbstractThe objective of this research is to fabricate injectable, polymeric composites that will act as scaffolds for bone ingrowth as well as carriers for the controlled release of bone growth factors. To that end, the injectable polyester poly(propylene fumarate) (PPF) was loaded with poly(DLlactic-co-glycolic acid) (PLGA) microparticles carrying the model drug FITC-dextran. This preparation was then crosslinked with N-vinyl pyrrolidinone in the presence of benzoyl peroxide as initiator and sodium chloride (NaCl) as leachable porogen. The encapsulation of growth factors in microparticles is necessary to minimize their denaturation during scaffold crosslinking. PLGA microparticles (0.04 g microparticles/g PPF) were incorporated into PPF composites having variable NaCl weight percents (50 and 70 wt% NaCl) and the effect on FITC-dextran release kinetics was determined in vitro for cylinders of diameter 6.5 mm and height 13.0 mm. The FITC-dextran loaded microparticles alone exhibited a large initial burst effect, while the composite materials displayed a smaller burst effect and a longer linear region of release. At day 3, 54.6±2.1%, 5.1±0.9%, and 12.5±0.3% of loaded FITC-dextran was released into pH 7.4 phosphate buffered saline from the microparticles, the 50 wt% NaCl, and the 70 wt% NaCl composites, respectively. By day 28, 90.9±6.9%, 12.7±1.7%, and 34.4±0.4% of loaded FITC-dextran was released. Our results demonstrate that PLGA microparticles can be incorporated into PPF composites and that the release kinetics of FITC-dextran can be systematically manipulated through alteration of the composite initial salt content.

Novel sustained-release dosage forms of proteins using polyglycerol esters of fatty acids

In order to develop a novel delivery system for proteins based on polyglycerol esters of fatty acids (PGEFs), we studied a model system using interferon-α (IFN-α) as the test protein. A cylindrical matrix was prepared by a heat extrusion technique using a lyophilized powder of the protein and 11 different types of synthetic PGEFs, which varied in degree of glycerol polymerization (di- and tetra-), chain length of fatty acids (myristate, palmitate and stearate) and degree of fatty acid esterification (mono-, di- and tri-). In an in-vitro release study using an enzyme-linked immunosorbent assay (ELISA) as a detection method, the matrices prepared from a monoglyceride (used for comparison) and from diglycerol esters exhibited a biphasic release pattern with a large initial burst followed by slow release. In contrast, the matrices prepared from tetraglycerol esters showed a steady rate of release without a large initial burst. In an in vivo release study, initial bursts of IFN-α release were, also, dramatically reduced when the matrices were prepared from the tetraglycerol esters of palmitate and stearate, and the mean residence time (MRT) of IFN-α was prolonged, whereas the matrices prepared from monoglyceride and from diglycerol esters showed large initial bursts of IFN-α release. Since the release rates from the matrices prepared from the tetraglycerol esters of palmitate and stearate were governed by Jander’s equation modified for a cylindrical matrix, the release from those matrices was concluded to be a diffusion-controlled process. The bioavailability of IFN-α after implantation of the matrix formulation prepared using all types of PGEFs, except for tetraglycerol triesters, was almost equivalent to that after injection of IFN-α solution; consequently, IFN-α in these matrices appears to remain stable during the release period.

Formation of elliptical galaxies at moderate redshifts

Different cosmological models make specific predictions about the number of elliptical galaxies as a function of redshift1, so observations can in principle be used to discriminate between those models. Traditionally, elliptical galaxies have been thought to have formed in a single, rapid burst of star formation at high redshifts ( z > 5), and then evolved quietly—with no significant further star formation—since that time2,3. Yet evidence suggests that at least some ellipticals formed for the merger of two spiral galaxies4,5. It remains unclear which process dominates the formation of elliptical galaxies. Here I use the results of deep optical6 and near-infrared7,8,9,10 images to show that there are fewer galaxies with very red colours than predicted by models in which typical ellipticals have completed their star formation by z ≈ 5, which means that elliptical galaxies must have significant star formation at z < 5. This requirement, combined with constraints on bursts of star formation in lower-redshift galaxies11,12, and the observed properties of galaxies in the redshift range 0< z < 1 (refs 13,14,15,16,17), suggests either that ellipticals form at moderate redshifts, where a large initial burst of star formation is shrouded by dust, or that they form through the merging of smaller galaxies.

Stabilization of 10-hydroxycamptothecin in poly(lactide-co-glycolide) microsphere delivery vehicles.

The purpose of this study was to investigate the potential of poly(lactide-co-glycolide) (PLGA) microspheres to stabilize and deliver the analogue of camptothecin, 10-hydroxycamptothecin (10-HCPT). 10-HCPT was encapsulated in PLGA 50:50 microspheres by using an oil-in-water emulsion-solvent evaporation method. The influence of encapsulation conditions (i.e., polymer molecular weight (Mw), polymer concentration, and carrier solvent composition) on the release of 10-HCPT from microspheres at 37 degrees C under perfect sink conditions was examined. Analysis of the drug stability in the microspheres was performed by two methods: i) by extraction of 10-HCPT from microspheres and ii) by sampling release media before lactone--carboxylate conversion could take place. Microspheres made of low Mw polymer (inherent viscosity 0.15 dl/g) exhibited more continuous drug release than those prepared from polymers of higher Mw (i.v. = 0.58 and 1.07 dl/g). In addition, a high polymer concentration and the presence of cosolvent in the carrier solution to dissolve 10-HCPT were both necessary in the microsphere preparation in order to eliminate a large initial burst of the released 10-HCPT. An optimal microsphere formulation released 10-HCPT slowly and continuously for over two months with a relatively small initial burst of the released drug. Both analytical methods used to assess the stability of 10-HCPT revealed that the unreleased camptothecin analogue in the microspheres remained in its active lactone form (> 95%) over the entire 2-month duration of study. PLGA carriers such as those described here may be clinically useful to stabilize and deliver camptothecins for the treatment of cancer.

Production of interleukin 1 from macrophages incubated with poly( dl-lactic acid) granules containing ovalbumin

The production profile of interleukin 1 (IL-1) from mouse peritoneal macrophages (Mø) was determined following their incubation with poly( dl-lactic acid) (PDLLA) granules containing ovalbumin (OVA). Upon incubation, Mø produced IL-1 at a significantly high rate compared with those incubated with OVA in the free form or OVA-free granules. A simple mixture of empty granules and free OVA exhibited the same level of IL-1 production as induced by free OVA alone. IL-1 production by the granules with a fixed OVA loading increased with an increase in their amount added to Mø. When incubated with a fixed amount of granules containing OVA of different loadings, Mø produced more IL-1 with an increase in the total OVA amount, but the IL-1 production decreased at OVA loadings higher than 10%. The presence of free OVA enhanced IL-1 production with the increased addition of empty granules, but the level induced by OVA loaded in granules was higher than that by mixtures of free OVA and empty granules, when compared at a similar OVA dose, irrespective of the absolute amount of PDLLA added. These findings indicate that the sustained release of OVA from the granules is critical to enhance the OVA-induced IL-1 production, in contrast to the OVA release accompanying a large initial burst, which reduced IL-1 production. It was concluded that the direct contact of PDLLA granules with Mø and the subsequent sustained release of OVA around Mø effectively activated Mø, resulting in enhanced IL-1 production.

Microencapsulation of ovalbumin in poly(lactide-co-glycolide) by an oil-in-oil (o/o) solvent evaporation method.

The objective of this study was to produce biodegradable poly(lactide-co-glycolide) (PLGA; 50/50) microspheres by an oil-in-oil (o/o) solvent evaporation method to prolong the in vitro release of ovalbumin (OVA) as a model protein. The effects, on loading efficiency, microsphere yield, morphology and drug release, of two dispersing agents, aluminium tristearate and Span 80, in mineral oil were examined. PLGA 50/50 microspheres containing OVA powder (sieved through a 53 microns mesh) were prepared using an o/o solvent evaporation method. When aluminum tristearate was employed as a dispersing agent, the loading efficiency and yield of OVA had maximum values of 89 and 72% at 0.15% (w/v) aluminum tristearate, respectively. Morphology studies suggested that the obtained microspheres were spherical, and had a smooth surface. The diameters of the microspheres ranged between 100 and 200 microns. The loading efficiency, or yield, for microspheres decreased significantly above or below 0.15% (w/v) aluminum tristearate, and microspheres with irregular shapes were observed. The minimum sedimentation volume ratio (F) was obtained at a dispersity of carbon black particles in ethanol containing 0.15% (w/v) aluminum tristearate by a sedimentation study, and the cloudy supernatant suggested a deflocculated suspension. However, on the contrary, when Span 80 was added into the mineral oil as a dispersing agent, the concentration of Span 80 had little or no effect on the characteristics of the prepared microspheres. Drug loadings (60-70%) were obtained within the Span 80 concentrations employed in the present study (0.05-1.0% (w/v)). The yields were also in the same levels. The microspheres prepared in mineral oil containing Span 80 had an average diameter less than 50 microns in all cases. Sustained-release characteristics were demonstrated for PLGA microspheres prepared in mineral oil containing aluminum tristearate as a dispersing agent, even though a burst release at the initial phase was observed. This initial burst release from PLGA microspheres was reduced to some extent by micronization of the OVA powder using a planetary-type ball mill. However, PLGA microspheres prepared in mineral oil containing Span 80 as a dispersing agent, exhibited a large initial burst release. This burst release seems to be due to the smaller size of microspheres and the OVA powder adhering to the surface of PLGA microspheres (confirmed by scanning electron microscope (SEM) study).

Enhanced antibody production through sustained antigen release from biodegradable granules

A proteinaceous model antigen, ovalbumin (OVA), was incorporated into poly(DL-lactic acid) (PDLLA) granules for sustained release of OVA over about 5 weeks without any large initial burst. When the granules were injected into the mouse peritoneal cavity, the production of anti-OVA IgG antibody in the serum was significantly enhanced and persisted for a long period compared with that induced by injection of OVA in the water-soluble form. Simple mixing of the PDLLA granules with free OVA had no enhancing effect on the antibody response, indicating that OVA incorporation in the granules is essential to enhance IgG production. The secondary response to antibody production was greater for mice given the primary immunization with the OVA-loaded granules than with free OVA. A body distribution study of OVA demonstrated that incorporation of OVA into PDLLA granules reduced its translocation to the blood circulation, leading to prolonged accumulation of OVA in the peritoneal cavity. Macrophages cocultivated with PDLLA granules containing OVA produced a larger amount of interleukin 1 than those cultured with other agents, suggesting that the PDLLA granules effectively activate the cell function for antibody production. It was concluded that the biodegradable granules were useful as a release carrier of antigens to enhance the antibody production.

Injectable Polyanhydride Granules Provide Controlled Release of Water-Soluble Drugs with a Reduced Initial Burst

A method for preparing polyanhydride granules of an injectable size was developed. The resulting granules permitted a nearly constant release of low-molecular-weight, water-soluble drugs without an initial burst. The polyanhydrides used were poly(fatty acid dimer), poly(sebacic acid), and their copolymers. The dyes acid orange 63 and p-nitroaniline were used as model compounds for drugs. Polymer degradation and drug release for disks and variously sized granules of copolymers containing drugs, prepared by a water-in-oil (W/O) emulsion method, were compared with those for devices prepared by the usual compression method. In the W/O emulsion method, a mixture of aqueous drug solution and polymer-chloroform solution was emulsified by probe sonication to prepare a very fine W/O emulsion. The powder obtained by freeze-drying of the W/O emulsion was pressed into circular disks. In the compression method, the drug was mechanically mixed with the polymer, and the mixture was compressed into circular disks. The resulting disks were ground to prepare granules of different sizes. The granules encapsulated more than 95% of the drug, irrespective of the preparation method. Both methods were effective in preparing polymer disks capable of controlled drug release without any initial burst. However, as the granule size decreased to an injectable size (diameter, < 150 microns), a large difference in the drug release profile was observed between the two preparation methods. The injectable granules obtained by the W/O emulsion method showed nearly constant drug release without any large initial burst, in contrast to those prepared by the compression method, irrespective of the drug type.(ABSTRACT TRUNCATED AT 250 WORDS)

A formulation method using D, L-lactic acid oligomer for protein release with reduced initial burst

This paper describes a preparation method of D,L-lactic acid oligomer formulations capable of sustained release of proteins without any large initial burst in release. The oligomer formulations containing various proteins were prepared by a W/O emulsion method to examine the protein release. Briefly, the W/O emulsion of protein aqueous solution and oligomer/chloroform solution was prepared by probe sonication. Then, the powders obtained by freeze-drying the W/O emulsion were compression molded at 60°C into rods. As another method (mixing method), the protein was mixed with the oligomer at 45–60°C and the mixture was molded into rods similarly. Moreover, the resulting rods were ground into granules. The recovery of the oligomer formulation was about 95% and the formulation incorporated about 90% of the theoretical amount of proteins, irrespective of the type of proteins and oligomers used. Both the W/O and mixing methods permitted sustained release of proteins from the oligomer rods without any large initial burst when the protein loading was less than 20%. However, the release profile was different between the granules prepared by the two methods. For granules of sizes less than 200μm prepared by the mixing method, greater than 50% of protein was released during the first day of the release test, irrespective of the oligomer molecular weight, the type of proteins, and their loading. On the contrary, the granules obtained by the emulsion method significantly reduced the extent of large initial burst in protein release. Light microscopic observations demonstrated a high homogeneity of protein dispersion through the oligomer formulation prepared by the W/O emulsion method, which led to the sustained release of proteins with a reduced initial burst. However, the emulsion method was associated with a loss in protein activity during preparation of the oligomer formulation. The emulsion method caused 45% and 63% loss in the biological activity of trypsin and insulin while about 10% of the activity was lost for the mixing method.

Controlled delivery systems for proteins using polyanhydride microspheres.

A method to provide near-constant sustained release of high molecular weight, water-soluble proteins from polyanhydride microspheres is described. The polyanhydrides used were poly(fatty acid dimer) (PFAD), poly(sebacic acid) (PSA), and their copolymers [P(FAD-SA)]. P(FAD-SA) microspheres containing proteins of different molecular sizes--lysozyme, trypsin, heparinase, ovalbumin, albumin, and immunoglobulin--were prepared by a solvent evaporation method using a double emulsion. The microspheres containing proteins were spherical, with diameters of 50-125 microns, and encapsulated more than 80% of the protein, irrespective of the protein used. Enzymatic activity studies showed that encapsulation of enzymes inside polyanhydride microspheres can protect them from activity loss. When not placed inside polyanhydride microspheres, trypsin lost 80% of its activity in solution at 37 degrees C at pH 7.4 in 12 hr, whereas inside the polyanhydride microspheres the activity loss was less than 10% under these conditions. About 47% of the enzymatic activity of heparinase encapsulated in the microspheres was lost at 37 degrees C in 24 hr, while in solution it lost over 90% of its activity. The protein-loaded microspheres displayed near-zero-order erosion kinetics over 5 days as judged by the release of sebacic acid (SA) from the microspheres. The microspheres degraded to form SA and FAD monomers. All proteins were released at a near-constant rate without any large initial burst, irrespective of polymer molecular weight and protein loading. The period of protein release was longer than that of SA and continued protein release was observed even after the microsphere matrix had completely degraded. Differential scanning calorimetric studies demonstrated an interaction between protein and the FAD monomers produced with microsphere degradation. It is likely that the protein interaction with FAD monomers permits formation of water-insoluble protein aggregates which slowly dissolve and diffuse out of the matrix, leading to delayed protein release. For trypsin-loaded microspheres, trypsin lost 40% of its activity during microsphere preparation. Activity studies demonstrated that the sonication process was primarily responsible for activity loss. A reduction in the period of ultrasound exposure decreased the loss of protein activity to around 20%.

Effects of counteranion of TRH and loading amount on control of TRH release from copoly(dl-lactic/glycolic acid) microspheres prepared by an in-water drying method

The preparation of injectable sustained release microspheres of TRH was investigated. The drug was encapsulated in copoly(dl-lactic/glycolic acid) (PLGA) using an in-water drying method through a w/o/w emulsion. The predominant factors influencing the entrapment ratio of TRH in the microspheres and the release rate of TRH from the microspheres were the loading amount and the dissociation state of the drug. Microspheres of TRH encapsulated as a free base at an adequate concentration provided a small initial burst followed by a zero-order prolonged release regardless of the high water solubility of the drug. The addition of strong acids to the inner aqueous phase interfered with the encapsulation efficacy and caused a large initial burst. The ionic interaction between the basic functional group of TRH and the carboxylic acid end group of PLGA during the preparation is necessary to produce a rigid matrix of the microspheres. This method is applicable to the microencapsulation of other water-soluble basic peptides in PLGA.