The influence of protein solubilisation, conformation and size on the burst release from poly(lactide-co-glycolide) microspheres (original) (raw)
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Journal of Controlled Release, 2001
Encapsulation of the model protein bovine serum albumin (BSA) into poly(D,L lactide-co-glycolide) (PLG) microspheres was performed by a non-aqueous oil-in-oil (o / o) methodology. Powder formulations of BSA obtained by spray-freeze drying were first suspended in methylene chloride containing PLG followed by coacervation by adding silicon oil and microsphere hardening in heptane. The secondary structure of BSA was determined at relevant steps of the encapsulation procedure by employing Fourier-transform infrared (FTIR) spectroscopy. This fast and non-invasive method demonstrated the potential to rapidly screen pharmaceutically relevant protein delivery systems for their suitability. Structural perturbations in BSA were reduced during the spray-freeze drying step by employing the excipient trehalose. The protein was then encapsulated into PLG microspheres under various conditions without inducing significant structural perturbations. BSA released from these microspheres had a similar monomer content as unencapsulated BSA and also the same secondary structure. Upon blending of a poloxamer (Pluronic F-68) with the polymer phase, in vitro release was characterized by a small initial release and a prolonged and continuous sustained phase. In conclusion, the developed o / o methodology coupled with FTIR spectroscopic monitoring of protein structure is a powerful approach for the development of sustained release microspheres.
Journal of Biomaterials Science-polymer Edition, 1997
Poly(ε-caprolactone) (PCL) microspheres containing c. 3% bovine serum albumin (BSA) were prepared by melt encapsulation and solvent evaporation techniques. PCL, because of its low Tm, enabled the melt encapsulation of BSA at 75°C thereby avoiding potentially toxic organic solvents such as dichloromethane (DCM). Unlike the solvent evaporation method, melt encapsulation led to 100% incorporation efficiency which is a key factor in the microencapsulation of water-soluble drugs. Examination of the stability of the encapsulated protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that protein integrity was unaffected by both methods of encapsulation. In vitro release of the protein into phosphate buffer examined at 37 °C from microspheres prepared by both techniques showed that the release rate from melt-encapsulated microspheres was somewhat slower compared to the release from solvent-evaporated spheres. Both released around 20% of the incorporated protein in 2 weeks amounting to approximately 6.5 µgmg-1 of microspheres. Although the diffusivity of macromolecules in PCL is rather low, it is shown that PCL microspheres are capable of delivering sufficient quantity of proteins by diffusion for prolonged periods to function as a carrier for many vaccines. Unlike poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) polymers which generate extreme acid environments during their degradation, the delayed degradation characteristics of PCL do not generate an acid environment during protein release and, therefore, may be advantageous for sustained delivery of proteins and polypeptides.
Journal of Pharmaceutical Investigation, 2012
Spray-dried microspheres based on polysaccharides were developed and the conformational stability and controlled release of incorporated protein were evaluated using bovine serum albumin (BSA) as a model protein. Microspheres composed of water soluble chitosan (WCS), hydroxypropyl-b-cyclodextrin (HP-b-CD) and polyethylene glycol (PEG) were prepared by spray dying. WCS was used as a mucoadhesive and biocompatible polymer. HP-b-CD and PEG were used as protein stabilizer during the spray drying process. Microspheres with 6-7 lm of mean diameter were successfully developed. Encapsulation efficiency of BSA in microsphere was over 70 %. Primary, secondary and tertiary structure of incorporated BSA in microsphere was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence intensity measurement, respectively. Conformational stability of BSA was maintained during the spray drying process. BSA release from microspheres was evaluated in in vitro model using the Transwell Ò insert, and showed a sustained release profile compared to naive BSA. Thus, these microspheres could possibly serve as an optimized delivery system for preserved stability and sustained release of protein.
Effect of additives on the release of a model protein from PLGA microspheres
AAPS PharmSciTech, 2001
The purpose of this study was to investigate the effect of 2 additives, poly(ethylene glycol) (PEG) 1000 and 1,2,3-tridecanoyl glycerol (tricaprin), on the physico-chemical characteristics and in vitro release of a model protein, bovine serum albumin (BSA), form poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. BSA-loaded microspheres were prepared by the double emulsion solvent evaporation method. Additives were incorporated into microspheres to modify the release of protein. The addition of PEG 1000 and tricaprin changed the surface characteristics of microspheres from smooth and nonporous to porous and dimpled, respectively. The in vitro release profiles showed that the additives significantly (P < 0.05) increased the early-stage release of BSA from microspheres.
Molecular Pharmaceutics, 2012
Polymer-based delivery systems provide a promising alternative to multidose intake of many drugs/vaccines. Protein aggregation and inactivation, however, are major problems associated with the encapsulation of proteins in microspheres. With this in mind, we investigated the structural integrity of a model protein bovine serum albumin (BSA) released from poly(lactide-co-glycolide) (PLGA) based microspheres. BSA was encapsulated using solid-in-oil-in-water (S/O/W) double emulsification method with different mixtures of surfactants (carboxymethyl cellulose (CMC):Tween 20/CMC:Tween 80/Tween 20:Tween 80) and with or without polyethylene glycol (PEG). The morphology of BSA-loaded microspheres was analyzed using dynamic light scattering (DLS) and scanning electron microscopy (SEM). BSA released from lyophilized microspheres was evaluated for the structural, conformational and thermal stability by using various spectroscopic and calorimetric techniques. Conformational analysis showed greater increase in secondary structural content of BSA in sample containing PEG and surfactant mixture of CMC and Tween 20 as compared to that containing other two mixtures of surfactants. The differential scanning calorimetric (DSC) analysis of released BSA from all PEG containing samples showed an increase in thermal stability of the protein. Furthermore, fluorescence spectra showed compactness of BSA. In conclusion our studies suggest macromolecular crowding, molecular confinement and increase in Gibbs free energy with strong electrostatic forces of repulsion between microspheres, the last phenomenon due to chosen surfactants, to be responsible for making the protein more compact and structurally integrated and result in a potential encapsulation process for improved protein integrity in final formulation.
Journal of Pharmacy and Pharmacology, 2001
Bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by a solid-in-oil-in-water (s/o/w) technique. We tested whether perturbations in BSA secondary structure could be minimized during encapsulation by using trehalose and how this would influence BSA aggregation and release. BSA secondary structure was monitored noninvasively by Fourier-transform infrared spectroscopy. When BSA was co-lyophilized with trehalose, lyophilization-induced structural perturbations were significantly reduced. The formulation obtained (BSA-Tre) was encapsulated into PLG microspheres and, by optimizing critical encapsulation parameters, a loading efficiency of 85 % was achieved. However, due to the loss of the excipient in the o/w emulsion step, the structure of BSA-Tre was more perturbed than before encapsulation. Excipient-loss and encapsulation-induced structural perturbations could be prevented by saturating the aqueous phase in the o/w step with trehalose and by using the organic solvent chloroform. This in turn reduced the formation of soluble BSA aggregates.
Journal of Controlled …, 1994
Poly (lactide-co-glycolide) microparticles, containing ricin toxoid or fluorescein isothiocyanate-labeled bovine serum albumin were prepared by a water-in-oil-in-water emulsion solvent extraction procedure with a high encapsulation efficiency (from 60% to 94%). Three agitation methods: vortex mixing, homogenization and sonication, were used to make the first inner w/o emulsion and the second w/o/w emulsion. The effects of process parameters on structure, surface condition, particle size, core loading and in vitro release properties of the protein-loaded microparticles were studied. SDS-PAGE analysis showed that none of the agitation methods damaged the structural integrity and stability of the encapsulated protein. Confocal laser scanning microscopic analysis and in vitro studies indicated that heterogeneous microparticles provided a fast release profile with a large protein burst (62%). and homogeneous microparticles released protein slower with a much lower protein burst (7%). These properties may influence the dynamics of the antibody response.
2004
The treatments for which proteins and peptides are prescribed as therapeutic agents require stable levels of active components over prolonged periods. One way to meet these requirements are sustained release systems, generally based on biodegradable polymers, of which polylactide (PLA) and its copolymers (PLGA) with glycolide (GA) are commonly used. An advantage of the lactide/glycolide copolymers is the well documented versatility in polymer properties (via manipulation of the comonomer ratio, molar mass, polymer crystallinity) and the corresponding performance characteristics (e.g., predictable in vivo degradation rates) (1, 2). In addition to the polymer chemistry, drug Poly(DL-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) copolymers were prepared by bulk ring opening polymerization of lactide and glycolide and characterized by GPC, FTIR, 1 H NMR and DSC. Copolymers with different molar masses at a constant lactide/glycolide ratio were used for preparation of bovine serum albumin (BSA)-loaded microparticles by the double emulsion w/o/w method. The influence of the copolymer molar mass and composition on the microparticle morphology, size, yield, degradation rate, BSA-loading efficiency and BSA release profile were studied. For microparticles prepared from PDLLGA copolymers, a biphasic profile for BSA release was found and for those made from PLLGA copolymers the release profile was typically triphasic; both of them were characterized by high initial burst release. Possible reasons for such behavior are discussed.
Journal of Pharmacy and Pharmacology, 2005
Poly(ethylene glycol) (PEG) was used as emulsifier to prepare α-chymotrypsin-loaded poly(lactic-coglycolic) acid (PLGA) microspheres by a solid-in-oil-in-water (s/o/w) technique. The effect of the molecular weight of PEG on protein stability was assessed by the determination of the amount of insoluble aggregates, the activity loss and the magnitude of structural perturbations. In addition, the effect of the molecular weight of PEG on the encapsulation efficiency, microsphere characteristics and release kinetics was investigated. X-ray photoelectron spectroscopy (XPS) was employed to characterize the surface chemistry of the microspheres. Microspheres were prepared using PEG with molecular weight of 6000, 8000, 10000, 12000 and 20000. The results indicate that PEG 20000 was the most effective emulsifier when producing α-chymotrypsin-loaded microspheres with respect to protein stability. The aggregate formation was decreased from 18% to 3%; the protein inactivation and the encapsulation-induced structural perturbations were largely prevented. XPS confirmed that PEG was largely located on the surface of microspheres. The molecular weight of PEG affected the microspheres' characteristics and release kinetics. Microspheres prepared with PEG 20000 showed improved encapsulation efficiency (80%) and a continuous release (for 50 days) with the lowest amount of initial release. It is demonstrated that the selection of the optimum molecular weight of PEG when used as emulsifier in the preparation of microspheres is a critical factor in the development of sustained-release formulations for the delivery of proteins.
International journal of pharmaceutics, 2002
When proteins are encapsulated in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation techniques, inactivation and aggregation are serious drawbacks hampering their sustained delivery. Hen egg-white lysozyme was employed to investigate whether stabilizing it towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of structurally unperturbed, non-aggregated, and active protein. When it was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres without stabilizing additives, lysozyme showed substantial loss in activity and aggregation. It has been shown that by co-dissolving various sugars and polyhydric alcohols with lysozyme in the first aqueous buffer, interface-induced lysozyme aggregation and inactivation can be minimized in the first emulsification step [J. Pharm. Pharmacol. 53 (2001) 1217]. Herein, it was found that those excipients, which were efficient in preventing interface-induced struct...