title: Annealing as a tool for the optimization of lyophilization and ensuring of the stability of protein-loaded PLGA nanoparticles.
abstract: The aim of this work was to use annealing as a tool to optimize the lyophilization cycle by decreasing its duration time, and simultaneously preserve the stability of poly(lactic-co-glycolic acid) nanoparticles and with upmost importance, maintain the structural stability of loaded insulin, used as model therapeutic protein. The contribution of a cryoprotectant in this preservation process was also evaluated. Insulin-loaded nanoparticles co-encapsulated with and without trehalose as cryoprotectant were produced, resulting in a particle size of about 250-300 nm, a PdI around 0.25 and a zeta potential in the range of -20 to -24 mV. The insulin association efficiency was higher than 90%, and the loading capacity was of 11-12%. The use of annealing allowed the decrease of duration time of primary drying in about 38%, representing a global decrease of lyophilization time of around 26%. The residual moisture content of all lyophilizates was around 1%, and the reconstitution of lyophilizates obtained using annealing was even faster than those without annealing. The co-encapsulated trehalose better preserved the nanoparticle size throughout the lyophilization process using annealing, compared to formulation containing no cryoprotectant. Fourier-transform infrared spectroscopy showed that the trehalose-containing nanoparticles presented higher insulin structural maintenance, compared to nanoparticles without cryoprotectant, presenting an insulin structural maintenance of 85.3 ± 0.7% and 86.0 ± 1.0% for annealing and no annealing, respectively. This formulation also presented the closest structural similarity with native insulin. Interestingly, the structural features of insulin loaded into nanoparticles upon lyophilization with and without annealing were practically identical, showing that annealing had no detrimental effect in insulin structure. Circular dichroism and fluorescence spectroscopy confirmed these results. Overall, this work gave rise to the importance of annealing in decreasing the duration time of lyophilization of protein-loaded poly(lactic-co-glycolic acid) nanoparticles, and simultaneously ensuring the stability of the carrier and loaded protein.
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; CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde and Instituto Universitário de Ciências da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra-Prd, Portugal. Electronic address: pedrofonte86@gmail.com.
authors.forename: Pedro
authors.initials: P
authors.lastname: Fonte
authors.affiliation: iMed.ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Paulo Roque
authors.initials: PR
authors.lastname: Lino
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde and Instituto Universitário de Ciências da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra-Prd, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Seabra
authors.affiliation: iMed.ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: António J
authors.initials: AJ
authors.lastname: Almeida
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 and Instituto Universitário de Ciências da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra-Prd, Portugal; i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; INEB-Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal. Electronic address: bruno.sarmento@ineb.up.pt.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D002626:Chemistry, Pharmaceutical; D002942:Circular Dichroism; D003451:Cryoprotective Agents / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry*; D005612:Freeze Drying; D007328:Insulin / Q000737:chemistry*; D019344:Lactic Acid / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D055550:Protein Stability; D017550:Spectroscopy, Fourier Transform Infrared; D014199:Trehalose / Q000737:chemistry
chemical_list: D003451:Cryoprotective Agents; D004337:Drug Carriers; D007328:Insulin; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D014199:Trehalose
keywords: Acetonitrile (PubChem CID: 6342); Annealing; Chloroform (PubChem CID: 6212); Dichloromethane (PubChem CID: 6344); FTIR; Insulin; Insulin (PubChem CID: 70678557); Lyophilization; Nanoparticle; PLGA; Poly(dl-lactic-co-glycolic acid) (PubChem CID: 23111554); Polyvinyl alcohol (PubChem CID: 11199); Protein; Trehalose (PubChem CID: 7427); Trifluoroacetic acid (PubChem CID: 6422)