title: Stability study perspective of the effect of freeze-drying using cryoprotectants on the structure of insulin loaded into PLGA nanoparticles.
abstract: This work aimed to evaluate the influence of a freeze-drying process using different cryoprotectants on the structure of insulin loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to assess the stability of these nanoparticles upon 6 months of storage following ICH guidelines. Insulin-loaded PLGA nanoparticles with a size around 450 nm were dehydrated using a standard freeze-drying cycle, using trehalose, glucose, sucrose, fructose, and sorbitol at 10% (w/v) as cryoprotectants. All formulations, except those nonadded of cryoprotectant and added with trehalose, collapsed after freeze-drying. The addition of cryoprotectants increased the nanoparticles stability upon storage. FTIR results showed that insulin maintained its structure after encapsulation in about 88%, decreasing to 71% after freeze-drying. The addition of cryoprotectants prior to freeze-drying increased insulin structural stability an average of up to 79%. Formulations collapsed after freeze-drying showed better protein stabilization upon storage, in special sorbitol added formulation, preserving 76, 80, and 78% of insulin structure at 4 °C, 25 °C/60% RH, and 40 °C/75% RH, respectively. Principal component analysis also showed that the sorbitol-added formulation showed the most similar insulin structural modifications among the tested storage conditions. These findings suggested that regarding nanoparticles stability, cryoprotectants are versatile to be used in a standard freeze-drying, however they present different performances on the stabilization of the loaded protein. Thus, on the freeze-drying of the nanoparticles field, this work gives rise to the importance of the process of optimization, searching for a balance between a good obtainable cake with an optimal structural stabilization of the loaded protein.
authors.affiliation: REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of Pharmacy, University of Porto , Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Fonte
authors.forename: Sandra
authors.initials: S
authors.lastname: Soares
authors.forename: Flávia
authors.initials: F
authors.lastname: Sousa
authors.forename: Ana
authors.initials: A
authors.lastname: Costa
authors.forename: Vítor
authors.initials: V
authors.lastname: Seabra
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods; D003451:Cryoprotective Agents / Q000737:chemistry*; D004355:Drug Stability; D005612:Freeze Drying / Q000379:methods; D005632:Fructose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D019344:Lactic Acid / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D013395:Sucrose / Q000737:chemistry; D014199:Trehalose / Q000737:chemistry
chemical_list: D003451:Cryoprotective Agents; D007328:Insulin; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D005632:Fructose; D019344:Lactic Acid; D013395:Sucrose; D014199:Trehalose; D005947:Glucose