Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

Research output: Contribution to journalJournal articleResearchpeer-review

  • Pedro Fonte
  • Fernanda Andrade
  • Cláudia Azevedo
  • João Pinto
  • Vítor Seabra
  • van de Weert, Marco
  • Salette Reis
  • Bruno Sarmento

PURPOSE: The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated.

METHODS: Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed.

RESULTS: Insulin was co-encapsulated with cryoprotectants resulting in particles of 243-394 nm, zeta potential of -32 to -35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA nanoparticles demonstrated enhanced hypoglycemic effect, comparatively to nanoparticles without cryoprotectant and added with trehalose, due to a superior insulin stabilization and bioactivity.

CONCLUSIONS: The freezing process may be detrimental to the structure of protein loaded into nanoparticles, with negative consequences to bioactivity. The co-encapsulation of cryoprotectants mitigated the freezing stresses with benefits to protein bioactivity.

Original languageEnglish
JournalPharmaceutical Research
Issue number11
Pages (from-to)2777–2793
Publication statusPublished - 2016

ID: 164467134