Encapsulation of lysozyme in a biodegradable polymer by precipitation with a vapor-over-liquid antisolvent (original) (raw)
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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...
Molecular Pharmaceutics, 2006
A new microencapsulation technique based on the solvent exchange method was implemented using an ultrasonic atomizer system to encapsulate a protein drug in mild conditions. The reservoir-type microcapsules encapsulating lysozyme as a model protein were prepared by inducing collisions between the aqueous droplets containing lysozyme and the droplets of organic solvent with dissolved poly(lactic acid-co-glycolic acid) (PLGA).The main focus of the study was to examine formulation variables on the size and the encapsulation efficiency of the formed microcapsules. The formulation variables examined were concentrations of mannose in the aqueous cores, NaCl in the aqueous collection medium, and PLGA in organic solvent. The mean diameter of the microcapsules ranged from 40 µm to 100 µm. Smaller microcapsules showed lower encapsulation efficiencies. The resulting microcapsules released native lysozyme in a sustained manner, and the release rate was dependent on the formulation conditions, such as the concentration and molecular weight of the polymer used. The solvent exchange method does not induce lysozyme aggregation and loss of its biological activity. The solvent exchange method, implemented by the ultrasonic atomizer system, provides an effective tool to prepare reservoir-type microcapsules for delivering proteins. . † Present address: Department of Advanced Polymer and Fiber Materials, Kyung Hee University, Gyeonggi-do 449-701, South Korea. (1) Bartus, R. T.; Tracy, M. A.; Emerich, D. F.; Zale, S. E. Sustained delivery of proteins for novel therapeutic agents. Science 1998, 281, 1161-1162. (2) Sinha, V. R.; Trehan, A. Biodegradable microspheres for protein delivery. J. Controlled Release 2003, 90, 261-280. (3) Tamber, H.; Johansen, P.; Merkle, H. P.; Gander, B. Formulation aspects of biodegradable polymeric microspheres for antigen delivery.
Biotechnology and Bioengineering, 2003
Encapsulation of proteins in poly(lactic-coglycolic) acid (PLGA) microspheres by the water-in-oil-inwater (w/o/w) technique is very challenging because of the inherent physical instability of proteins. In particular, exposure of proteins to the first water-in-oil emulsion causes unwanted interface-induced protein inactivation and aggregation. We tested whether salts could afford stabilization of a model protein, hen egg-white lysozyme, against the detrimental events occurring at the w/o interface and subsequently upon w/o/w encapsulation. First, we investigated the effect of salts on the specific enzyme activity and generation of soluble precipitates and insoluble aggregates upon emulsification of an aqueous lysozyme solution with methylene chloride. It was found that lysozyme precipitation occurred upon emulsification. The amount of precipitate formed at salt concentrations between 10-100 mM was related to the position of the anion in the electroselectivity series (SO 4 2− > SCN − > Cl − > H 2 PO 4 − ) while high salt concentrations (1M) led to > 80% of lysozyme precipitation regardless of the salt. The precipitates consisted of buffer-soluble protein precipitates and water-insoluble noncovalent aggregates. Lysozyme precipitation, aggregation, and inactivation upon emulsification were largely prevented in the presence of 50 mM KH 2 PO 4 while KSCN caused an increase in these detrimental events. Second, it was tested whether the improved structural integrity of lysozyme at the w/o interface would improve its stability upon w/o/w encapsulation in PLGA microspheres. Some conditions indeed led to improved stability, particularly codissolving lysozyme with 50 mM KH 2 PO 4 reduced loss in the specific activity and aggregation. In conclusion, the type and concentration of salts is a critical parameter when encapsulating protein in PLGA microspheres.
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.
Using complex coacervation for lysozyme encapsulation by spray-drying
Journal of Food Engineering, 2016
The purpose of this study is to evaluate the properties of encapsulated lysozyme (at 0.714 g/L concentration) by spray-drying as well as the effect of complexation with increased pectin concentrations (0-4 g/L) on the preservation of lysozyme structure and activity. Maximum turbidity of spray-dried complexes was lower than unprocessed ones at a threshold pectin concentration above which turbidity was reduced. This obtained result could be attributed to the formation of more individual complexes with decreased aggregation upon application of spray-drying due to configurational changes of the complexes. At higher pectin concentrations (above 0.5 g/L), spray-dried complexes exhibited lower antimicrobial activity compared with unprocessed complexes probably because of high limitation in diffusion and accessibility of substrate to active sites of the encapsulated enzyme within the polysaccharide network. Conformational changes of lysozyme and formation of complexes with pectin upon spray-drying were determined by using fluorescence and UV-Vis absorption measurements. The results revealed that spray-drying had an apparent a significant effect on lysozyme structure, activity, and mobility particularly at higher pectin concentrations (above 0.5 g/L).
Compressed CO 2 antisolvent precipitation of lysozyme
Journal of Supercritical Fluids, 2009
The precipitation of proteins using compressed CO 2 as antisolvent in the precipitation with a compressed antisolvent (PCA) process has been investigated for lysozyme in its DMSO solution. In the vapor liquid coexistence region of the solvent-antisolvent mixture, particles between 100 and 200 nm were obtained, whereas at supercritical and liquid conditions studied in the 25-40 • C and 100-150 bar ranges, particle sizes were reproducibly below 100 nm. No effect on particle size was observed for variations of the CO 2 and solutions flow rates between 40 and 120 g/min, and between 0.4 and 2 ml/min, respectively. Phase behavior experiments indicate that protein precipitation results from a metastable liquid-liquid phase separation, with the protein rich phase remaining trapped in a gel-like state. Moreover, we suggest that due to the high CO 2 concentrations involved in PCA, phase separation takes place by spinodal decomposition. The proposed precipitation mechanism is not limited to the PCA process, but can be extended also to results obtained for the precipitation of lysozyme using the gas antisolvent (GAS) process.
Iranian journal of pharmaceutical research : IJPR, 2011
Lysozyme, as a model protein, was precipitated through the formation of protein-Zn complex to micronize for subsequent encapsulation within poly (lactic-co-glycolic acid) (PLGA) microspheres. Various parameters, including pH, type and concentration of added salts and protein concentration, were modified to optimize the yield of protein complexation and precipitation. The resulting protein particles (lysozyme-Zn complex as a freshly prepared suspension or a freeze-dried solid) were then loaded into PLGA (Resomer(®) 503H) microspheres, using a double emulsion technique and microspheres encapsulation efficiency and their sizes were determined. It was observed that salt type could significantly influence the magnitude of protein complexation. At the same conditions, zinc chloride was found to be more successful in producing pelletizable lysozyme. Generally, higher concentrations of protein solution led also to the higher yields of complexation and at the optimum conditions, the percenta...
Controlled Release of Lysozyme from Double-Walled Poly(Lactide-Co-Glycolide) (PLGA) Microspheres
Polymers
Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w 1 /o/o/w 2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme.
Journal of Controlled Release, 2005
Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for a-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a h-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.