Release Profiles Of Bovine Serum Albumin Research Articles

Stability of bovine serum albumin complexed with PEG-poly( l-histidine) diblock copolymer in PLGA microspheres

The aim of this study was to examine the stability of bovine serum albumin (BSA) in poly( dl-lactic acid- co-glycolic acid) (PLGA) microspheres upon addition of a new excipient, poly(ethylene glycol)-poly( l-histidine) diblock copolymer (PEG-PH). Poly( l-histidine) component can form an ionic complex with BSA under acidic conditions within a narrow pH range. To optimize the ionic complexation conditions for BSA with PEG-PH, the resulting complex sizes were monitored using the Zetasizer. PLGA microspheres containing BSA as a model protein were prepared by w/o/w double emulsion method. BSA stability in aqueous solutions and after release from PLGA microspheres was determined using circular dichroism (CD) spectroscopy for secondary structure analyses and fluorescence measurements for tertiary structure analyses. The release profile of BSA from the microspheres was monitored using UV spectrophotometry. The rate of PLGA degradation was monitored by gel permeation chromatography. The pH profile within microspheres was further evaluated by confocal microscopy using a pH-sensitive dye. Approximately 19 PEG-PH molecules and one BSA molecule coalesced to form an ionic complex around a pH range of 5.0–6.0. Plain BSA/PLGA and BSA/PEG-PH/PLGA microspheres had a mean size of 27–35 μm. PLGA microspheres with a BSA loading efficiency > 80% were prepared using the double emulsion method. PEG-PH significantly improved the stability of BSA both in aqueous solutions and in PLGA microspheres. The release profiles of BSA from different formulations of PLGA microspheres were significantly different. PEG-PH effectively buffered the local acidity inside the microspheres and improved BSA release kinetics by reducing initial burst release and extending continuous release over a period of time, when encapsulated as an ionic complex. PLGA degradation rate was found to be delayed by PEG-PH. There was clear evidence that PEG-PH played multiple roles when complexed with BSA and incorporated into PLGA microspheres. PEG-PH is an effective excipient for preserving the structural stability of BSA in aqueous solution and BSA/PLGA microspheres formulation.

Effect of Osmotic Pressure in the Solvent Extraction Phase on BSA Release Profile from PLGA Microspheres

This study investigated the influence of osmotic pressure in the organic solvent extraction phase on release profile of bovine serum albumin (BSA) from poly(lactide-co-glycolide) (PLGA) microspheres. BSA-loaded PLGA microspheres with a target load of 10% were prepared by a double emulsion phase separation method. All the microsphere batches were fabricated in the same conditions except that in the organic solvent (CH2Cl2) evaporation step. Different concentrations of NaCl (0, 1.8, and 3.6%) or sucrose (20%) were used to generate a range of osmotic pressures in the extraction aqueous phase. These microspheres were characterized for incorporation efficiency, surface and internal morphology, particle size, protein stability, and in vitro release. The microspheres were spherical with particle size ranging from 16.8 to 27.8 μm. Higher osmotic pressure resulted in a denser internal structure although similar nonporous surface morphology was observed with all batches. No significant difference in encapsulation efficiency existed from batch to batch (87–94%). Sodium dodecyl sulfate–polyamide gel electrophoresis showed that BSA integrity was well retained. The release profile of the batch prepared with only water as the continuous (solvent extraction) phase exhibited a 79% burst release in the first 24 hr followed by a plateau and then a little release after 21 days. In the presence of NaCl or sucrose, the burst effect significantly decreased with increase in osmotic pressure in the extraction aqueous phase, which was then followed by sustained release for 35 days. A mass balance was made when the release terminated. Therefore, in the organic solvent extraction and evaporation step, increasing the osmotic pressure in the aqueous phase both reduced the burst release from the microspheres and improved the subsequent sustained release profile.

Effect of polymer compositions on the fabrication of poly(ortho‐ester) microspheres for controlled release of protein

AbstractEight kinds of self‐catalyzed poly(ortho‐esters) (POEs) are used to fabricate bovine serum albumin (BSA)‐containing microspheres using a W/O/W double‐emulsion solvent extraction/evaporation method. All eight kinds of POE polymers used in this study are shown able to form microspheres under proposed fabrication conditions. The surface morphology and inner structure of the microspheres are analyzed using scanning electron microscopy (SEM). The microspheres have a size range from 64.7 to 120.2 μm. POE with a higher viscosity leads to bigger microspheres. It was found that the POE composition has a significant effect on BSA release profiles. POEs, which are more hydrophilic and contain a greater amount of glycolide or lactate (latent acid), yield higher BSA release rates. Specifically, POE containing 1,6‐hexanediol diglycolide (HD‐diGL) microspheres have the highest BSA release rate after a 20‐day test through a combination of surface erosion and diffusion mechanisms. POE containing a high percentage of the trans‐cyclohexanedimethanol (CDM) segment tends to yield microspheres with a lower release rate because of its hydrophobic nature. It was also found that the BSA release rate is more rapid at 37°C than at 22°C because of faster polymer degradation and water penetration at 37°C. Experimental results suggest that various protein release rates can be achieved by using different compositions of POEs. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 1630–1642, 2001

Applicability of various amphiphilic polymers to the modification of protein release kinetics from biodegradable reservoir-type microspheres

The effects of various amphiphilic polymers on the kinetics of protein release from reservoir-type microspheres, prepared by a solid-in-oil-in-water emulsion-solvent evaporation method, were investigated. Bovine serum albumin (BSA), as a model protein, was firstly micronized through co-lyophilization with amphiphilic polymers, such as poly (ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), and pluronic F68. This process was based on the aqueous phase separation of protein and amphiphilic polymer induced by freezing-condensation. Mixing of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) (at a ratio of 4:6) in a methylene chloride solution provided a‘polymer-alloy’ structure, where the preformed solid BSA microparticles were selectively distributed in the inner PLGA-rich phase. The reservoir-type microspheres obtained through this process showed high entrapment efficiencies (more than 85%) and reduced initial burst releases (less than 10%). Although PVP did not modify the BSA release profile, PEG and pluronic F68 enhanced the BSA release, with no increase of the initial burst effect, responding to their loading percentage: 3% loading of PEG or pluronic F68 resulted in typical zero-order release kinetics. The abilities of these amphiphilic polymers to modify the protein release profile could be predicted from their partitioning characteristics in the polymer-alloys and in the methylene chloride/water system.

Effects of inner water volume on the peculiar surface morphology of microspheres fabricated by double emulsion technique

Poly(D,L-lactide-co-glycolide) (PLG, 65:35) was used to encapsulate bovine serum albumin (BSA) using a water-in-oil-in-water (W/O/W) double emulsion solvent extraction technique. To investigate the effects of an inner water/oil ratio on microsphere characteristics, microspheres were fabricated using four different formulations with a fixed oil volume of 12 ml and the inner aqueous phase volume of 0.2 ml, 0.3 ml, 0.4 ml or 0.5 ml, respectively. Spherical microspheres were obtained after collection by filtration for formulations employing any of the four different inner water/oil ratios. However, microspheres with smaller inner water volumes tend to collapse after vacuum drying. The surface of the formulation with a higher inner water/oil ratio was shown to possess many more pores than that of the formulations with lower inner water/ oil ratios. These pores may facilitate the water withdrawal during vacuum drying. Furthermore, microspheres with the lowest inner water/oil ratio (1/60) had higher initial burst release due to its larger surface area. However, microspheres with the highest inner water volume yield a faster release profile of BSA due to interconnected voids within microspheres and more pores on the surface. Therefore, the inner water/oil ratio is a crucial factor in the W/O/W double emulsion technique affecting the morphology and release kinetics of the resulting microspheres.

Diffusional characteristics of freeze/thawed poly(vinyl alcohol) hydrogels: Applications to protein controlled release from multilaminate devices

The incorporation of a model protein, bovine serum albumin (BSA), into poly(vinyl alcohol) (PVA) hydrogel films during the freezing and thawing process and its subsequent release behavior were investigated. The effect of the number of freezing and thawing cycles as well as the stability of BSA were examined. BSA release profiles were not significantly different from gels prepared after 3 or 5 cycles. However, the rate and overall amount of PVA dissolution were considerably higher for gels prepared after 3 cycles. These observations were then applied to the development of novel, freeze/thawed PVA laminates. Laminates containing gel layers prepared after 3 or 5 cycles were successfully prepared with good stability over a 6 month swelling period. These structures, containing distinct layers of very specific properties, could be used to achieve zero-order release behavior.

Stereocomplex formation between enantiomeric PLA-PEG-PLA triblock copolymers: Characterization and use as protein-delivery microparticulate carriers

Two enantiomeric triblock ABA copolymers composed of poly(L-lactide)–poly(ethylene glycol)–poly(L-lactide) (PLLA–PEG–PLLA) and poly(D-lactide)–poly(ethylene glycol)–poly(D-lactide) (PDLA–PEG–PDLA) were synthesized with two different middle-block PEG chain lengths by ring-opening polymerization of L-lactide and D-lactide in the presence of PEG, respectively. A pair of enantiomeric triblock copolymers were combined to form a stereocomplex by a solvent-casting method. The triblock copolymers and their stereocomplexes were characterized by 1H- and 13C-NMR spectroscopy and gel permeation chromatography. Their crystalline structures and crystalline melting behaviors were analyzed by the wide-angle X-ray diffraction method and differential scanning calorimetry. The stereocomplex formed between a pair of enantiomeric triblock copolymers exhibited a higher crystalline melting temperature with a distinctive 3/1 helical crystalline structure. PLLA–PEG–PLLA and its stereocomplex with PDLA–PEG–PDLA were used to fabricate a series of microspheres encapsulating a model protein drug, bovine serum albumin (BSA). They were prepared by a double-emulsion solvent-evaporation method. The morphological aspects of the microspheres were characterized and BSA release profiles from them were investigated. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1615–1623, 2000

Protein-loaded poly(epsilon-caprolactone) microparticles. I. Optimization of the preparation by (water-in-oil)-in water emulsion solvent evaporation

The aim of this work was to optimize protein entrapment in pure poly(epsiloncaprolactone) (PCL) microparticles (MP) using the (water-in-oil)-in water solvent evaporation technique and bovine serum albumin (BSA) as drug model. Therefore, the preparative variables such as polymer solvent, protein/polymer ratio, polymer molecular weight, internal aqueous/organic phases ratio, organic/ external aqueous phase ratio, and nature of the emulsifier were evaluated on microparticle characteristics such as BSA entrapment, entrapment efficiency, size and morphology. The in vitro release profiles of BSA from such MP in two different media with or without sodium dodecyl sulphate (SDS) were investigated. In optimum conditions, smooth and spherical pure PCL MP with high encapsulation efficiency (50.29=5.01%) were prepared. The release profiles of BSA in the release media were significantly different and faster in the presence of SDS. Moreover, they exhibited a relatively low burst effect after 24 h (<30%) followed by a continuous release over 28 days. Due to PCL's numerous desirable characteristics, such MP could be an exciting alternative for the controlled release of proteinaceous compounds.

Bovine serum albumin release from poly(α-hydroxy acid) microspheres: effects of polymer molecular weight and surface properties

Two commercially available PLGA polymers (lactic acid/glycolic acid 50:50, with respective molecular weights of 87 and 15 kDa) were used to prepare microspheres containing a model protein, bovine serum albumin (BSA), following a process based on the (water-in-oil)-in-water emulsion solvent evaporation/extraction technique. The influence of the formulation parameters on the in vitro release profiles of BSA was assessed. The results showed that both the encapsulation efficiency and the protein release rate were closely dependent on the molecular weight of the coating polymer. The increase of the encapsulation efficiency, and the quasi-absence of burst effect with the low molecular weight polymer, are explained by ionic and physical interactions between the protein and the wall polymer. In addition, contact angle measurements revealed the absence of protein at the surface of the microspheres and showed that the burst effect, measured with the highest molecular weight polymer, could not be explained by desorption of the protein from the surface but rather by the diffusion of the protein from regions bordering the microsphere surface.

Biodegradable microcapsules prepared by a w/o/w technique: effects of shear force to make a primary w/o emulsion on their morphology and protein release.

A water-in-oil-in-water (w/o/w) technique, sometimes known as in-water drying method, was used to prepare microcapsules consisting of polylactic acid and poly(lactide-co-glycolide). The influence of shear force to produce an initial water-in-oil (w/o) emulsion on the characteristics of microcapsules and protein release was investigated. Bovine serum albumin (BSA) was used as the model protein drug for encapsulation. The initial w/o emulsion was prepared by a Polytron homogenizer. The shear rate was varied from 11 to 23 krpm to produce w/o emulsions with different shear forces. This study revealed pronounced effects of shear force on the characteristics of microcapsules and release profiles of BSA. Depending on the degree of the shear applied, the inner structure of microcapsules showed very different morphology, which was responsible for different release patterns. A low shear produced microcapsules with a high initial burst release of BSA, whereas microcapsules using a high shear exhibited a controlled release of protein without any initial burst release. Also, at a given shear force, a variation in polymer composition of microcapsules was found to be effective in controlling the release characteristics of protein. Thus, the homogenization technique should be carefully considered in designing microcapsules with desirable release profiles of proteins and an adequate period of protein delivery.

The influence of biodegradable microcapsule formulations on the controlled release of a protein

Biodegradable microcapsules were prepared from various polymer compositions such as poly- d,l-lactic acid or poly- l-lactic acid and poly- d,l-lactide-co-glycolide for the controlled release of a model protein Bovine Serum Albumin (BSA). ELISA data indicated that the binding activity of BSA toward anti-BSA antibody was not lost during the microencapsulation process and the release from microcapsules. The effect of different microcapsule formulations on release profiles of the protein was investigated. It was found that the polymer composition, total amount of polymers and protein loading were factors involved in the determination of release profiles of BSA. The degradation of various microcapsules was also investigated. Factors such as degrees of the hydrolytic chain scission of ester linkages, water content and internal structures were compared. The microcapsule formulation influenced the porosity and the degree of concentration gradient of BSA. These were related to the initial burst effects and release patterns. All these factors interplayed their roles on the release characteristics of BSA. Depending on the microcapsule formulation, controllable and predictable release profiles of BSA that could be described by either zero- or firstorder kinetics were observed.

Evaluation of cholesterol-lecithin implants for sustained delivery of antigen: release in vivo and single-step immunisation of mice

Bovine serum albumin (BSA), a model antigen, was delivered from matrix systems (pellets) prepared from cholesterol (C) alone, and from C and hydrogenated egg lecithin (PC) in various C-PC ratios, to mice by implanting the pellets subcutaneously. The in vivo release profile of BSA from these pellets and the antibody responses to BSA were studied at intervals over a period of 10 months following a single-dose administration. An initial burst release of BSA was observed within 2 days of implantation followed by a trickle delivery over 7 months. During the first 5 days, the rate of release of BSA from C pellets (without PC) was significantly slower than from C-PC pellets of 4:1 and 8:1 ratios ( w w ). At day 9, the differences became insignificant. The concentration of BSA retained in the pellets increased with an increase in PC content of the pellets. The implants containing BSA induced anti-BSA antibodies at 2 months and maintained the same levels of antibodies for up to 10 months. BSA delivered in the form of three injections produced the same level of antibodies as C-PC pellets of 1:2 ratio ( w w ), but a significantly lower level than that produced by the C pellets and C-PC pellets of 1:1, 2:1, 4:1 and 8:1 ratios ( w w ) ( P < 0.04). Bioerosion of the C-PC pellets (at ≦ 4:1, w w ) was noticeable and an increase in PC content of the pellets increased the erosion rate.

Cholesterol and lecithin implants for sustained release of antigen: release and erosion in vitro, and antibody response in mice

Implantable matrix systems (pellets) were prepared from cholesterol (C), and from C and lecithin (PC), with bovine serum albumin (BSA) as a model antigen. The release profile of BSA from these pellets and their erosion behaviour in borate buffer (pH 7.4) were studied in vitro under different hydrodynamic conditions. The effect of different methods used to mix C and PC on release profile and erosion behaviour was also investigated; pellets were made from physically mixed and coprecipitated C and PC. Ethanol, ethanol-chloroform (1:1, v/v) and chloroform were used as coprecipitating solvents. BSA was released by diffusion from all pellets and increasing the concentration of PC enhanced the rate of release. The erosion rate of C and PC depended on the C-PC composition of pellets. Both the release of BSA and erosion of pellets were positively influenced by hydrodynamic activities. At a C-PC ratio of 1:2 w/w (approx. 1 : 1 molar), a paracrystalline hydrated phase (gel) was formed which was not a diffusional barrier for the release of BSA. Chloroform used as a coprecipitating solvent reduced the rate of release of BSA significantly at C-PC ratios of 2:1 and 1:1 (w/w). Erosion of pellets occurred when implanted subcutaneously in mice for 40 days. Implantation of mice with the different formulations containing BSA induced significant ( P ≦ 0.0001) antibodies to BSA in all groups at 40 days, but the levels did not differ significantly between groups (P ≧ 0.088).