title: Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization.
abstract: 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.
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-113, Porto, Portugal. pedrofonte86@gmail.com.|CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116, Gandra PRD, Portugal. pedrofonte86@gmail.com.
authors.forename: Pedro
authors.identifier: 0000-0002-1115-9282
authors.initials: P
authors.lastname: Fonte
authors.affiliation: Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-113, Porto, Portugal.|IBEC, Institute for Bioengineering of Catalonia, 08028, Barcelona, Spain.|School of Pharmacy, University of Barcelona, 08028, Barcelona, Spain.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Andrade
authors.affiliation: i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.|INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Azevedo
authors.affiliation: iMed. ULisboa, Department of Galenic Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal.
authors.forename: João
authors.initials: J
authors.lastname: Pinto
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116, Gandra PRD, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Seabra
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-113, Porto, Portugal.
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116, Gandra PRD, Portugal. bruno.sarmento@ineb.up.pt.|i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. bruno.sarmento@ineb.up.pt.|INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal. bruno.sarmento@ineb.up.pt.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D000818:Animals; D003451:Cryoprotective Agents / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry; D065546:Drug Liberation; D004355:Drug Stability; D005612:Freeze Drying / Q000379:methods*; D005615:Freezing; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000494:pharmacology*; D007328:Insulin / Q000737:chemistry / Q000494:pharmacology*; D019344:Lactic Acid / Q000737:chemistry*; D008297:Male; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D017208:Rats, Wistar; D013395:Sucrose / Q000737:chemistry; D013499:Surface Properties; D014199:Trehalose / Q000737:chemistry
chemical_list: D003451:Cryoprotective Agents; D004337:Drug Carriers; D007004:Hypoglycemic Agents; D007328:Insulin; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D013395:Sucrose; D014199:Trehalose
keywords: PLGA nanoparticles; cryoprotectant; freezing; insulin; lyophilization