Effect of salts on lysozyme stability at the water–oil interface and upon encapsulation in poly(lactic-co-glycolic) acid microspheres (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...
J Pharm Pharmacol, 2001
Protein inactivation and aggregation at the water/CH 2 Cl 2 interface is one of the most detrimental events hindering the encapsulation of structurally unperturbed proteins into poly(lactide-co-glycolide) (PLG) microspheres for their clinical application as sustained release dosage forms. We have investigated the inactivation and aggregation of the model protein hen egg-white lysozyme at this interface and devised methods to prevent both events. When lysozyme was exposed to a large water/CH 2 Cl 2 interface achieved by homogenization, lysozyme aggregation occurred. Fourier-transform infrared (FTIR) spectroscopic data demonstrated that the aggregates formed contained intermolecular β-sheets. The aggregates were of a noncovalent nature because they slowly dissolved in D 2 O and the IR spectral bands typical for the intermolecular β-sheets disappeared at approximately 1617 and 1690 cm − 1 . The observed loss in specific enzyme activity of soluble lysozyme was caused by the irreversible formation of an unfolded lysozyme species, which was found to be monomeric, and was able to leave the water/CH 2 Cl 2 interface and accumulate in the aqueous phase. Polyols were, in a concentration dependent fashion, efficient in ameliorating lysozyme unfolding and aggregation. However, prevention of lysozyme aggregation and activity loss in the various samples were unrelated.
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.
Journal of Pharmacy and Pharmacology, 2001
Protein inactivation and aggregation at the water/CH 2 Cl 2 interface is one of the most detrimental events hindering the encapsulation of structurally unperturbed proteins into poly(lactide-co-glycolide) (PLG) microspheres for their clinical application as sustained release dosage forms. We have investigated the inactivation and aggregation of the model protein hen egg-white lysozyme at this interface and devised methods to prevent both events. When lysozyme was exposed to a large water/CH 2 Cl 2 interface achieved by homogenization, lysozyme aggregation occurred. Fourier-transform infrared (FTIR) spectroscopic data demonstrated that the aggregates formed contained intermolecular β-sheets. The aggregates were of a noncovalent nature because they slowly dissolved in D 2 O and the IR spectral bands typical for the intermolecular β-sheets disappeared at approximately 1617 and 1690 cm − 1 . The observed loss in specific enzyme activity of soluble lysozyme was caused by the irreversible formation of an unfolded lysozyme species, which was found to be monomeric, and was able to leave the water/CH 2 Cl 2 interface and accumulate in the aqueous phase. Polyols were, in a concentration dependent fashion, efficient in ameliorating lysozyme unfolding and aggregation. However, prevention of lysozyme aggregation and activity loss in the various samples were unrelated.
Journal of Pharmaceutical Sciences, 1999
0 Lysozyme was encapsulated in biodegradable polymer microspheres which were precipitated from an organic solution by spraying the solution into carbon dioxide. The polymer, either poly-(l-lactide) (l-PLA) or poly(DL-lactide-co-glycolide) (PGLA), in dichloromethane solution with suspended lysozyme was sprayed into a CO 2 vapor phase through a capillary nozzle to form droplets which solidified after falling into a CO 2 liquid phase. By delaying precipitation in the vapor phase, the primary particles became sufficiently large, from 5 to 70 µm, such that they could encapsulate the lysozyme. At an optimal temperature of −20°C, the polymer solution mixed rapidly with CO 2 , and the precipitated primary particles were sufficiently hard such that agglomeration was markedly reduced compared with higher temperatures. More uniform particles were formed by flowing CO 2 at high velocity in a coaxial nozzle to mix the droplets at the CO 2 vapor− liquid interface. This process offers a means to produce encapsulated proteins in poly(DL-lactide-co-glycolide) microspheres without earlier limitations of massive polymer agglomeration and limited protein solubility in organic solvents. .Figure 1sSchematic of the apparatus for precipitation with a vapor-overliquid compressed fluid antisolvent.
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...
Influence of some formulation parameters on lysozyme adsorption and on its stability in solution
International journal of pharmaceutics, 2002
According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.
European Journal of Pharmaceutics and Biopharmaceutics, 2010
Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a twodimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.
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.