Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation (original) (raw)
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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 Pharmacy and Pharmacology, 2001
Non-aqueous protocols to encapsulate pharmaceutical proteins into biocompatible polymers have gained much attention because they allow for the minimization of procedure-induced protein structural perturbations. The aim of this study was to determine if these advantages could be extended to a semi-aqueous encapsulation procedure, namely the solid-in-oil-inwater (s/o/w) technique. The model protein bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by first suspending lyophilized BSA in methylene chloride containing PLG, followed by emulsification in a 1 % aqueous solution of poly(vinyl alcohol). By variation of critical encapsulation parameters (homogenization intensity, BSA :PLG ratio, emulsifier concentration, ratio of organic to aqueous phase) an encapsulation efficiency of 90 % was achieved. The microspheres obtained showed an initial burst release of 20 %, a sustained release over a period of about 19 days, and a cumulative release of at least 90 % of the encapsulated BSA. Different release profiles were observed when using different encapsulation protocols. These differences were related to differences in the microsphere erosion observed using scanning electron microscopy. Release of BSA was mainly due to simple diffusion or to both diffusion and microsphere erosion. Fourier-transform infrared studies were conducted to investigate the secondary structure of BSA during the encapsulation. Quantification of the α-helix and β-sheet content as well as of overall structural changes showed that the secondary structure of encapsulated BSA was not more perturbed than in the lyophilized powder used initially. Thus, the encapsulation procedure did not cause detrimental structural perturbations in BSA. In summary, the results demonstrate that the s/o/w technique is an excellent alternative to the water-in-oil-in-water technique, which is still mainly used in the encapsulation of proteins in PLG microspheres.
Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications
Journal of Controlled Release, 2012
Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight -Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C 60 . Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.
Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein
Brazilian Journal of Pharmaceutical Sciences, 2020
Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactideco-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.
Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres
European Journal of Pharmaceutics and Biopharmaceutics, 2008
Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, a-chymotrypsin, peroxidase and b-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.
Pharmaceutical Research, 2008
Purpose. To evaluate the real-time effects of formulation and instrumental variables on microcapsule formation via natural jet segmentation, a new microencapsulation system termed the microenvironmentcontrolled encapsulation (MiCE) process was developed. Methods. A modified flow cytometer nozzle hydrodynamically focuses an inner drug and outer polymer solution emanating from a coaxial needle assembly into a two-layer compound jet. Poly(lacticco-glycolic acid) (PLGA) dissolved in a water-miscible organic solvent resulted in formation of reservoir-type microcapsules by interfacial phase separation induced at the boundary between the PLGA solution and aqueous sheath. Results. The MiCE process produced microcapsules with mean diameters ranging from 15-25 mm. The resultant microcapsule size distribution and number of drug cores existing within each microcapsule was largely influenced by the PLGA concentration and microcapsule collection method. Higher PLGA concentrations yielded higher mean diameters of single-core microcapsules. Higher drug solution flow rates increased the core size, while higher PLGA solution flow rates increased the PLGA film thickness. Conclusion. The MiCE microencapsulation process allows effective monitoring and control of the instrumental parameters affecting microcapsule production. However, the microcapsule collection method in this process needs to be further optimized to obtain microcapsules with desired morphologies, precise membrane thicknesses, high encapsulation efficiencies, and tight size distributions.
Development of polylactide microspheres for protein encapsulation and delivery
Journal of Applied Polymer Science, 2002
The development of injectable microparticles for protein delivery is a major challenge. We demonstrated the possibility of entrapping human serum albumin (HSA) and thrombin (Thr) in poly(ethylene glycol) (PEG)-coated, monodisperse, biodegradable microspheres with a mean diameter of about 10 m. In our earlier studies, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) analysis was used to characterize the surface of PEG-coated, taxol-loaded poly(lactic acid) (PLA) microspheres. An analysis by DRIFTS revealed that PEG was incorporated well on the PLA microsphere surface. An emulsion of protein (in water) and PLA dissolved in an acetone-dichloromethane (or acetone-chloroform) mixture were poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA)] with stirring with a high-speed homogenizer for the formation of microparticles. HSA recovery in microspheres ranged from 13 to 40%, depending on the solvent and emulsification systems used for the preparation. PLA dissolved in a dichloromethane/acetone system and albumin loaded via a PEG emulsification solution (PLA-PEG-HSA) showed maximum drug recovery (39.5%) and drug content (9.9%). Scanning electron microscopy revealed that PEG-coated microspheres had less surface micropores than PVA-based preparations. The drug-release behavior of microspheres suspended in phosphate-buffered saline exhibited a biphasic pattern. An initial burst release (30%) followed by a constant slow release for 20 days was observed for HSA and Thr from PLA-PEG microspheres. PEG-coated PLA microspheres show great potential for protein-based drug delivery.
A method to tune the shape of protein-encapsulated polymeric microspheres
Scientific Reports, 2015
Protein encapsulation technologies of polymeric microspheres currently in use have been optimized to effectively protect their "protein cargo" from inactivation occurring in biological environments, preserving its bioactivity during release up to several weeks. The scenario of protein delivery would greatly benefit by strategies enabling the production of non-spherical particles. Herein we report an easy and effective stamp-based method to produce poly-lactic-glycolic-acid (PLGA) microparticles encapsulating Vascular Endothelial Growth Factor (VEGF) of different shapes. We demonstrate that PLGA microspheres can be deformed at room temperature exploiting solvent/non-solvent plasticization in order to preserve the properties of the starting microspheres. This gentle method allows the production of shaped particles that provide a prolonged release of VEGF in active form, as verified by an angiogenic assay. The retention of the biological activity of an extremely labile molecule, i.e. VEGF, lets us hypothesize that a wide variety of drug and protein encapsulated polymeric microspheres can be processed using this method.
Discussion and outlook 145 Curriculum Vitae 149 Publications 149 [2] A. Smith, I.M. Hunneyball, Evaluation of poly(lactic acid) as a biodegrad¬ able drug delivery system for parenteral administration, Int. J. Pharm. 30 (1986)215-220. Background and purpose 3 [3] J.M. Anderson, M.S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres, Adv. Drug. contribution to over¬ coming the problem of residual solvents in biodegradable microspheres prepared by coacervation, Eur. irradiation for terminal sterilization of 17ß-estradiol loaded poly-(D.L-lactide-co-glycolide) microspheres, J. Control. Release 61 (1999) 203-217. [7] A. Hausberger, R. Kenley, P.P. De Luca, Gamma irradiation effects on molecular weight and in vitro degradation of poly(D,L-lactide-coglycolide) microparticles, Pharm. Res. 12 (1995) 851-856. 4 Background and purpose Abstract 5 Abstract
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.