Improved activity and stability of lysozyme at the water/CH2CI2 interface: enzyme unfolding and aggregation and its prevention by polyols

Pérez, Caroline; Griebenow, Kai

doi:10.1211/0022357011776667

Cited by 52 publications

(30 citation statements)

References 25 publications

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“…While a self-protecting effect was observed for lysozyme, it was difficult to prevent aggregation at such high concentrations. An 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/methylene chloride interface and accumulate in the aqueous phase (55).…”

Section: Protein Instability During Plg Depot Loadingmentioning

confidence: 99%

Poly(ethylene glycol)-Modified Proteins: Implications for Poly(lactide-co-glycolide)-Based Microsphere Delivery

Pai

Tilton

Przybycien

2009

AAPS J

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Abstract. The reduced injection frequency and more nearly constant serum concentrations afforded by sustained release devices have been exploited for the chronic delivery of several therapeutic peptides via poly(lactide-co-glycolide) (PLG) microspheres. The clinical success of these formulations has motivated the exploration of similar depot systems for chronic protein delivery; however, this application has not been fully realized in practice. Problems with the delivery of unmodified proteins in PLG depot systems include high initial "burst" release and irreversible adsorption of protein to the biodegradable polymer. Further, protein activity may be lost due to the damaging effects of protein-interface and protein-surface interactions that occur during both microsphere formation and release. Several techniques are discussed in this review that may improve the performance of PLG depot delivery systems for proteins. One promising approach is the covalent attachment of poly(ethylene glycol) (PEG) to the protein prior to encapsulation in the PLG microspheres. The combination of the extended circulation time of PEGylated proteins and the shielding and potential stabilizing effects of the attached PEG may lead to improved release kinetics from PLG microsphere system and more complete release of the active conjugate.

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Section: Protein Instability During Plg Depot Loadingmentioning

confidence: 99%

Poly(ethylene glycol)-Modified Proteins: Implications for Poly(lactide-co-glycolide)-Based Microsphere Delivery

Pai

Tilton

Przybycien

2009

AAPS J

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“…Recovery of soluble lysozyme after emulsification was determined by measuring the protein concentration before and after sample treatment as reported previously (Pérez and Griebenow, 2001). …”

Section: Lysozyme Recoverymentioning

confidence: 99%

“…The protein concentration was used to determine the actual protein loading of the PLGA microspheres (%, w/w). The actual loading was used to calculate the encapsulation efficiency (Pérez et al, 2001). …”

Section: Protein Loading Of Microspheresmentioning

confidence: 99%

“…Formation of the first emulsion in the w/o/w technique is the main cause for protein inactivation and aggregation (Alonso et al, 1994;Lu and Park, 1995;Morlock et al, 1997;Nihant et al, 1994;Pérez and Griebenow, 2001;Pérez et al, 2002aPérez et al, , 2002bSah, 1999aSah, , 1999bSah, , 1999cVan de Weert et al, 2000). The tremendous stability challenges are best demonstrated when very stable single-subunit single-domain proteins are encapsulated.…”

Section: Introductionmentioning

confidence: 99%

“…The tremendous stability challenges are best demonstrated when very stable single-subunit single-domain proteins are encapsulated. For example, a significant fraction of the thermodynamically very stable protein hen egg-white lysozyme was inactivated and formed noncovalent aggregates when an aqueous protein solution was emulsified with methylene chloride (Pérez and Griebenow, 2001; Van de Weert et al, 2000), the solvent most commonly employed in w/o/w encapsulation of proteins. Lysozyme aggregation and inactivation was reduced by codissolving sugars (i.e., lactulose) in the first aqueous phase.…”

Section: Introductionmentioning

confidence: 99%

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Effect of salts on lysozyme stability at the water–oil interface and upon encapsulation in poly(lactic‐co‐glycolic) acid microspheres

Pérez

Griebenow

2003

Biotech & Bioengineering

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Encapsulation of proteins in poly(lactic-co-glycolic) acid (PLGA) microspheres by the water-in-oil-in-water (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.

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Preparation and properties of crosslinked chitosan thermosensitive hydrogel for injectable drug delivery systems

Zan

Chen

Jiang

et al. 2006

J of Applied Polymer Sci

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ABSTRACT:The aim of this study was to prepare and investigate the physical properties of a thermosensitive crosslinked chitosan pregel solution, and evaluate the in vitro release profiles of macromolecules from this sol-gel transition system. Chitosan and poly (vinyl alcohol) were used to form an interpenetrating polymeric network with glutaraldehyde as the crosslinker, and glycerophosphate (GP) was added to transform the pH-dependent solutions into thermosensitive pH-dependent solutions. Rheological study showed that the gelation was dependent on the crosslink degree and GP concentration of the solution. The crosslinked gel had excellent mechanic properties and no apparent "pores" and formed an integrated hydrogel texture according to scanning electronic micrograph. Gas chromatography test guaranteed the medication safety with no detection of glutaraldehyde remnants in the hydrogels. In vitro release study showed that the gelation does not significantly affect the macromolecules diffusion but the crosslinking degree does. These results indicated that the hydrogel formed an intensified three-dimensional hybrid network with interpenetrating molecules, which effectively buffered or delayed the macromolecules diffusion. The hydrogels sustained the drug release over 30 days and could be potentially used as in situ gelling implants.

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Improved activity and stability of lysozyme at the water/CH2CI2 interface: enzyme unfolding and aggregation and its prevention by polyols

Cited by 52 publications

References 25 publications

Poly(ethylene glycol)-Modified Proteins: Implications for Poly(lactide-co-glycolide)-Based Microsphere Delivery

Poly(ethylene glycol)-Modified Proteins: Implications for Poly(lactide-co-glycolide)-Based Microsphere Delivery

Effect of salts on lysozyme stability at the water–oil interface and upon encapsulation in poly(lactic‐co‐glycolic) acid microspheres

Preparation and properties of crosslinked chitosan thermosensitive hydrogel for injectable drug delivery systems

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