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The potential of cyclodextrins to stabilize alpha-chymotrypsin upon encapsulation in Poly(lactic-co-glycolic) acid (PLGA) microspheres using a solid-in-oil-in-water (s/o/w) technique was investigated. Two cyclodextrins, hydroxyl-propyl-beta-cyclodextrin (HPbetaCD) and methyl-beta-cyclodextrin (MbetaCD), one insoluble and the other soluble in methylene chloride, were used. The results demonstrate that HPbetaCD failed to stabilize alpha-chymotrypsin upon encapsulation. Specifically, 19% of the protein was aggregated and the specific activity of the enzyme was reduced to ca. 50% of that prior to encapsulation. In contrast, MbetaCD significantly decreased the formation of aggregates to 3% and the retained specific activity of the enzyme was approximately 90%. The co-lyophilization of alpha-chymotrypsin with MbetaCD prior to encapsulation was a requisite to preserve the protein stability in microspheres. Furthermore, MbetaCD prevented the loss of protein during the preparation of microspheres and the encapsulation efficiency was improved to 90%. Release experiments showed the use of MbetaCD modified the release profile: the burst release decreased from 54% (in the absence of the excipient) to 36%. The results suggest that MbetaCD might be a suitable excipient to improve protein stability in s/o/w encapsulation procedures.

Poly(ethylene glycol) (PEG) was used as emulsifier to prepare alpha-chymotrypsin-loaded poly(lactic-co-glycolic) 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 6,000, 8,000, 10,000, 12,000 and 20,000. The results indicate that PEG 20,000 was the most effective emulsifier when producing alpha-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 20,000 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.

The effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. alpha-Chymotrypsin was chemically modified with PEG (M(w) = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrypsin molar ratios between approximately 1 and 8 were considered because higher levels of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation., (Copyright 2004 Wiley-Liss, Inc.)

Purpose: To investigate the potential of different solvents with better biocompatibility to replace CH2Cl2 in the encapsulation of alpha-chymotrypsin in poly (lactic-co-glycolic) acid (PLGA) microspheres without causing protein instability., Methods: The oil-to-water (O:W) ratio in the emulsification step of the solid-in-oil-in-water (s/o/w) encapsulation process was optimized with respect to maximizing protein stability and encapsulation efficiency for various solvents. Formation of insoluble aggregates and residual enzyme activity were primarily used as stability parameters. Several solvents possessing low toxicity with different water solubility were used to prepare alpha-chymotrypsin loaded PLGA microspheres., Results: The O:W ratio in the emulsification step is critical with respect to maintaining protein stability. This was related to the solvents' water solubility. In general, hydrophilic solvents were detrimental to protein stability and encapsulation efficiency. However, after optimization of the O:W ratio for solvents with different water solubility, protein stability was preserved during encapsulation using butyl acetate when poly (ethylene glycol) (PEG) was used as the emulsifying agent (ca. 1% of non-covalent aggregates and 93 +/- 10% of residual specific activity)., Conclusions: The s/o/w technique was successfully improved by replacing the ICH class 2 solvent CH2Cl2 with the class 3 solvent butyl acetate without compromising alpha-chymotrypsin stability.

Development of alternative procedures to the commonly employed water-in-oil-in-water technique to encapsulate proteins in polymers is needed due to protein stability issues. Herein the model protein gamma-chymotrypsin has been encapsulated in poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres using the solid-in-oil-in-water (s/o/w) encapsulation technique. The model protein was chosen because it has a measurable biological activity and its unfolding is irreversible. The latter make the protein an excellent sensor for unfolding events in the encapsulation procedure. While lyophilization did not cause any irreversible aggregation or loss in activity, encapsulation of the lyophilized enzyme by the s/o/w technique proved detrimental to its integrity. Specifically, 34% of the encapsulated protein was aggregated and the specific activity of enzyme released within 24 h was reduced to ca. 50% of that prior to encapsulation. FTIR spectra demonstrated substantial encapsulation-induced perturbations of the secondary structure of gamma-chymotrypsin. To achieve stabilization of gamma-chymotrypsin during encapsulation, excipients were employed during the initial lyophilization process. When gamma-chymotrypsin was co-lyophilized with poly(ethylene glycol) (PEG) the formation of non-covalent aggregates inside the microspheres decreased significantly to 8%. FTIR data showed that PEG prevented encapsulation-induced structural perturbations. In contrast, the amount of aggregates remained high (34%) when gamma-chymotrypsin was co-lyophilized with trehalose. No additional non-soluble aggregates were formed during 1 week of in vitro release. Furthermore, the amount of non-soluble aggregates in the microspheres after encapsulation correlated with the amount of non-released protein. Therefore in vitro release did not cause aggregation. Similar results were found with respect to the retention of the specific enzyme activity where PEG afforded excellent stability.