Pharmaceutical Research

, Volume 33, Issue 11, pp 2777–2793 | Cite as

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

  • Pedro FonteEmail author
  • Fernanda Andrade
  • Cláudia Azevedo
  • João Pinto
  • Vítor Seabra
  • Marco van de Weert
  • Salette Reis
  • Bruno SarmentoEmail author
Research Paper



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.


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.


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.


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.


cryoprotectant freezing insulin lyophilization PLGA nanoparticles 



Area above the curve


Association efficiency


Attenuated Total Reflectance-Fourier transform infrared spectroscopy


Circular dichroism


Differential scanning calorimetry


High performance liquid chromatography


Loading capacity


Pharmacological availability


Poly(lactic-co-glycolic acid)


Polyvinyl alcohol


Retention efficiency


Scanning electron microscopy


Collapse temperature


Glass transition temperature


Glass transition temperature of the frozen sample


X-Ray powder diffraction



Pedro Fonte and Fernanda Andrade would like to thank Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/SAL-FCT/104492/2008; SFRH/BD/78127/2011) and (SFRH/BD/73062/2010) for financial support. This work was also financed by European Regional Development Fund (ERDF) through the Programa Operacional Factores de Competitividade − COMPETE, by Portuguese funds through FCT in the framework of the project PEst-C/SAU/LA0002/2013, and cofinanced by North Portugal Regional Operational Programme (ON.2 − O Novo Norte) in the framework of Project SAESCTN-PIIC&DT/2011 under the National Strategic Reference Framework (NSRF). It is also acknowledged the financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013 - POCI/01/0145/FERDER/007728). Abbot Laboratories, Portugal is acknowledged for kindly provide the Precision Xtra® blood glucose meter and test strips. The authors also thank to Dorthe Ørbæk from the Faculty of Health and Medical Sciences, University of Copenhagen, for the XRPD experiments.

Supplementary material

11095_2016_2004_MOESM1_ESM.docx (561 kb)
ESM 1 (DOCX 561 kb)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.UCIBIO, REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of PharmacyUniversity of PortoPortoPortugal
  2. 2.CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da SaúdeGandra PRDPortugal
  3. 3.Laboratory of Pharmaceutical Technology, Faculty of PharmacyUniversity of PortoPortoPortugal
  4. 4.IBEC, Institute for Bioengineering of CataloniaBarcelonaSpain
  5. 5.School of PharmacyUniversity of BarcelonaBarcelonaSpain
  6. 6.i3S, Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  7. 7.INEB - Instituto de Engenharia BiomédicaUniversidade do PortoPortoPortugal
  8. 8.iMed. ULisboa, Department of Galenic Pharmacy and Pharmaceutical Technology, Faculty of PharmacyUniversity of LisbonLisbonPortugal
  9. 9.Department of Pharmacy, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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