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Extraction of PLGA-Microencapsulated Proteins Using a Two-Immiscible Liquid Phases System Containing Surfactants

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Abstract

Purpose

The extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated.

Methods

First, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry.

Results

Without surfactants, protein recovery was only 27–43% for IFN-α2b and 58–73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins.

Conclusion

Proteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

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Abbreviations

BCA:

bicinchoninic acid

EGF:

epidermal growth factor

ELISA:

enzyme-linked immunosorbent assay

ESI-MS:

electrospray ionization mass spectrometry

IFN:

interferon

mAb:

monoclonal antibody

PLA:

poly (L-lactide)

PLGA:

poly (DL-lactide-co-glycolide)

PVA:

polyvinyl alcohol

RP-HPLC:

reversed-phase high performance liquid chromatography

SDS-PAGE:

sodium dodecylsulfate-polyacrylamide gel electrophoresis

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Acknowledgments and Disclosures

We would like to thank MSc. Haydee Gerónimo, Olga Brito and Joaquín González for EGF and IFN-α2b biological activity assays. We are also grateful to MSc. Yanet Terrero for the RP-HPLC analysis of EGF samples. The authors really appreciate the invaluable help of BSc. Yosep Rodríguez, MSc. Jesús Noda and Dr. Vladimir Besada for performing the ESI-MS analyses.

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Authors and Affiliations

  1. Formulation Development Department Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba

    Vivian Saez, Liurdis Caballero, Raymersy Aldana, Elián Cruz & Rolando Paez

  2. Macromolecular Chemistry Department Biomaterials Center (BIOMAT), University of Havana, P.O. Box 6130, Havana, Cuba

    José A. Ramón & Carlos Peniche

  3. Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana 6, CP 10600, Cuba

    Vivian Saez

Authors
  1. Vivian Saez
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  2. José A. Ramón
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  3. Liurdis Caballero
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  7. Rolando Paez
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Correspondence to Vivian Saez.

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Saez, V., Ramón, J.A., Caballero, L. et al. Extraction of PLGA-Microencapsulated Proteins Using a Two-Immiscible Liquid Phases System Containing Surfactants. Pharm Res 30, 606–615 (2013). https://doi.org/10.1007/s11095-012-0916-0

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  • DOI: https://doi.org/10.1007/s11095-012-0916-0

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