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Improved α-Chymotrypsin Stability Upon Encapsulation in PLGA Microspheres by Solvent Replacement

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Abstract

Purpose. To investigate the potential of different solvents with better biocompatibility to replace CH2Cl2 in the encapsulation of α-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 α-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 α-chymotrypsin stability.

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References

  1. H. Sah. Microencapsulation techniques using ethyl acetate as a dispersed solvent: effects of its extraction rate on the characteristics of PLGA microspheres. J. Control. Release 47:233-245 (1997).

    Google Scholar 

  2. S. W. Cho, S. H. Song, and Y. W. Choi. Effects of solvent selection and fabrication method on the characteristics of biodegradable poly(lactide-co-glycolide) microspheres containing ovalbumin. Arch. Pharm. Res. 23:385-390 (2000).

    Google Scholar 

  3. J. Herrman and R. Bodmeier. Somatostatin containing biodegradable microspheres prepared by a modified solvent evaporation method based on W/O/W-multiple emulsions. Int. J. Pharm. 126:129-138 (1995).

    Google Scholar 

  4. D. T. Birnbaum, J. D. Kosmala, D. B. Henthorn, and L. Brannon-Peppas. Controlled release of β-estradiol from PLAGA microparticles: The effect of organic phase solvent on encapsulation and release. J. Control. Release 65:375-387 (2000).

    Google Scholar 

  5. J. P. McGee, S. S. Davis, and D. T. O'Hagan. Zero order release of protein from poly(D,L-lactide-co-glycolide) microparticles prepared using a modified phase separation technique. J. Control. Release 34:77-86 (1995).

    Google Scholar 

  6. K. S. Soppimath and T. M. Aminabhavi. Ethyl acetate as a dispersing solvent in the production of poly(DL-lactide-co-glycolide) microspheres: effect of process parameters and polymer type. J. Microencapsulation 19:281-292 (2002).

    Google Scholar 

  7. I. J. Castellanos, W. L. Cuadrado, and K. Griebenow. Prevention of structural perturbations and aggregation upon encapsulation of bovine serum albumin into poly(lactide-co-glycolide) microspheres using the solid-in-oil-in-water technique. J. Pharm. Pharmacol. 53:099-1107 (2001).

    Google Scholar 

  8. P. Johansen, Y. Men, R. Audran, G. Corradin, H. P. Merkle, and B. Gander. Improving stability of microencapsulated tetanus toxoid by co-encapsulation of additives. Pharm. Res. 15:1103-1110 (1998).

    Google Scholar 

  9. M. J. Blanco-Príeto, K. Besseghir, O. Zerbe, D. Andris, P. Orsolini, F. Heimgartner, H. P. Merkle, and B. Gander. In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres. J. Control. Release 67:19-28 (2000).

    Google Scholar 

  10. H. Sah. Ethyl formate-alternative dispersed solvent useful in preparing PLGA microspheres. Int. J. Pharm. 195:103-113 (2000).

    Google Scholar 

  11. K. S. Soppimath, A. R. Kulkarni, and T. M. Aminabhavi. Development of hollow microspheres as floating controlled release systems for cardiovascular drugs: preparation and release characteristics. Drug Dev. Ind. Pharm. 27:507-515 (2001).

    Google Scholar 

  12. H. Sah, M. S. Smith, and R. T. Chern. A novel method of preparing PLGA microcapsules utilizing methylethyl ketone. Pharm. Res. 13:360-367 (1996).

    Google Scholar 

  13. W. Al-Azzam, E. A. Pastrana, and K. Griebenow. Co-lyophilization of bovine serum albumin (BSA) with poly(ethylene glycol) improves efficiency of BSA encapsulation and stability in polyester microspheres by a solid-in-oil-in-oil-technique. Biotechnol. Lett. 24:1367-1374 (2002).

    Google Scholar 

  14. T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, and H. Yoshino. Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant. J. Controlled Release 69:434-444 (2000).

    Google Scholar 

  15. I. J. Castellanos, K. G. Carrasquillo, J. D. Lopez, M. Alvarez, and K. Griebenow. Encapsulation of bovine serum albumin in poly(lactide-co-glycolide) microspheres by the solid-in-oil-in-water technique. J. Pharm. Pharmacol. 53:167-178 (2001).

    Google Scholar 

  16. I. J. Castellanos, G. Cruz, R. Crespo, and K. Griebenow. Encapsulation-induced aggregation and loss in activity of γ-chymotrypsin and their prevention. J. Control. Release 81:307-319 (2002).

    Google Scholar 

  17. E. G. DelMar, C. Laigman, J. W. Brodrick, and M. C. Geokas. A sensitive new substrate for chymotrypsin. Anal. Biochem. 99:316-320 (1979).

    Google Scholar 

  18. K. G. Carrasquillo, R. A. Cordero, S. Ho, J. M. Franquiz, and K. Griebenow. Structure-guided encapsulation of bovine serum albumin in poly(DL-lactic-co-glycolic)acid. Pharm. Pharmacol. Commun 4:563-571 (1998).

    Google Scholar 

  19. K. G. Carrasquillo, H. R. Constantino, R. A. Cordero, C. C. Shu, and K. Griebenow. On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer. J. Pharm. Sci. 88:166-173 (1999).

    Google Scholar 

  20. K. Griebenow and A. M. Klibanov. On protein denaturation in aqueous-organic but not in pure organic solvents. J. Am. Chem. Soc. 118:11695-11700 (1996).

    Google Scholar 

  21. K. G. Carrasquillo, C. Sanchez, and K. Griebenow. Relationship between conformational stability and lyophilization-induced structural changes in chymotrypsin. Biotechnol. Appl. Biochem. 31:41-53 (2000).

    Google Scholar 

  22. S. J. Prestrelski, N. Tedishi, T. Arakawa, and J. F. Carpenter. Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. Biophys. J. 65:661-671 (1993).

    Google Scholar 

  23. I. J. Castellanos, R. Crespo, and K. Griebenow. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J. Control. Release 81:307-319 (2003).

    Google Scholar 

  24. R. Bodmeier and J. W. McGinity. Solvent selection in the preparation of poly(DL-lactide) microspheres prepared by the solvent evaporation method. Int. J. Pharm. 43:179-186 (1988).

    Google Scholar 

  25. A. Hickel, C. J. Radke, and H. W. Blanch. Role of the organic solvents on Pa-hydroxynitrile lyase interfacial activity and stability. Biotechnol. Bioeng. 74:18-28 (2001).

    Google Scholar 

  26. J. M. Péan, F. Boury, M. Venier-Julienne, P. Menei, J. Proust, and J. P. Benoit. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres? Pharm. Res. 16:1294-1299 (1999).

    Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, University of Puerto Rico, Río Piedras Campus, PO Box 23346, San Juan, PR 00931-3346, USA

    Ingrid J. Castellanos

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  1. Ingrid J. Castellanos
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  2. Kai Griebenow
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Castellanos, I.J., Griebenow, K. Improved α-Chymotrypsin Stability Upon Encapsulation in PLGA Microspheres by Solvent Replacement. Pharm Res 20, 1873–1880 (2003). https://doi.org/10.1023/B:PHAM.0000003388.59659.fa

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  • DOI: https://doi.org/10.1023/B:PHAM.0000003388.59659.fa

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