title: Effect of freeze-drying, cryoprotectants and storage conditions on the stability of secondary structure of insulin-loaded solid lipid nanoparticles.
abstract: This study aims to monitor the secondary structure behaviour of insulin when it is encapsulated into solid lipid nanoparticles (SLN), under the influence of several critical processing parameters. Insulin was used as a therapeutic protein model. Physicochemical properties of insulin-loaded SLN (Ins-SLN) were assessed, with special focus on the insulin secondary structure after its encapsulation into SLN and after freeze-drying using different cryoprotectants (glucose, fructose and sorbitol). Additionally, a 6-month stability study was performed to evaluate the maintenance of insulin secondary structure over time at different storage conditions (4 °C/60% RH, 25 °C/60% RH, 40 °C/75% RH). Ins-SLN were successfully produced with a mean and narrow particle size around 400 nm, zeta potential around -13 mV, an insulin association efficiency of 84%. Physical-chemical properties of SLN were maintained after freeze-drying. FTIR results showed that encapsulated insulin maintained a native-like structure in a degree of similarity around 92% after production, and 84% after freeze-drying. After 6 months, freeze-dried Ins-SLN without cryoprotectant stored at 40 °C/75% RH presented the same degree of structure preservation and morphology. Results revealed that insulin structure can be significantly protected by SLN matrix itself, without a cryoprotectant agent, even using a non-optimized freeze-drying process, and under the harsher storage conditions. Multivariable experimental settled the process parameters to fit with the desired product quality attributes regarding protein and nanoparticle stability.
authors.affiliation: Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde - Norte, CESPU, Gandra-PRD, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Soares
authors.forename: Pedro
authors.initials: P
authors.lastname: Fonte
authors.forename: Ana
authors.initials: A
authors.lastname: Costa
authors.forename: José
authors.initials: J
authors.lastname: Andrade
authors.forename: Vítor
authors.initials: V
authors.lastname: Seabra
authors.forename: Domingos
authors.initials: D
authors.lastname: Ferreira
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D003451:Cryoprotective Agents / Q000737:chemistry*; D004355:Drug Stability; D004356:Drug Storage / Q000379:methods; D005612:Freeze Drying / Q000379:methods*; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size
chemical_list: D003451:Cryoprotective Agents; D007328:Insulin; D008055:Lipids
keywords: Cryoprotectants; FTIR; Freeze-drying; Glyceryl behenate (PubChem CID: 5362585); Glyceryl palmitostearate (PubChem CID: 114690); Insulin secondary structure; Polyvinyl alcohol (PubChem CID: 11199); Solid lipid nanoparticles; Stability study; Tween 80 (PubChem CID: 5281955).; d-Fructose (PubChem CID: 5984); d-Glucose (PubChem CID: 5793); d-Sorbitol (PubChem CID: 5780)