The aim of this research was to explore the possibility of using two different production technologies, such as Spray Congealing (SC) and Spray Freeze Drying (SFD) in the preparation of Solid Lipid Microparticles (SLMs) containing proteins. Bovine Serum Albumin (BSA) was employed as model protein due to its well-known physical-chemical state characterization. SC technology is based on spraying a fluid consisting of a solution or suspension of active pharmaceutical ingredient in a molten carrier, which melts at a relatively low temperature (50-80°C). Conversely, in SFD process, a solution or a biphasic system containing the dissolved protein is sprayed into a cryogenic liquid (-70°C) to form droplets which freeze upon the contact with the cryogenic solution and this suspension is then freeze dried to obtain a fine powder. In the first case BSA might be denaturated by heat, while in latter case, the degrading action could result from both the use of solvents and the freeze-drying process. Six batches of microparticles employing three lipid (Precirol®ATO 5, Dynasan®114 and Dynasan®118) with different melting temperatures and containing 10% and 20 % of BSA were produced by SC. On the contrary, using SFD technology , two formulations containing 9% and 16% of BSA in the presence of Dynasan®114 as lipid carrier, lecithin and poloxamer 407 as surfactants and sucrose as lyoprotector were preparedt. The results showed process yields ranging between 65% to 80% and encapsulation efficiencies above 90% and between 80% and 90% for SC and SFD formulations, respectively. SEM analysis displayed that all batches produced by SC were spherical and not aggregated with a mean volume diameter ranging between 150-300 micron. Conversely, SFD formulations showed numerous surface fractures, which appeared or as small cracks or as noticeable lacks of lipid membrane. Physical-chemical characterization using differential scanning calorimetry (DSC), UV spectrophotometry in fourth derivative mode and Raman microspectroscopy was performed. Thermal analysis showed that the protein degradation in solid state started around 80°C. UV spectrophotometry, however, highlighted that no spectral variations occurred between aqueous solution of BSA (reference) and BSA solutions obtained by hot extraction (70-75°C) of the SLMs produced by both techniques. The Raman microspectroscopy, associated with the curve fitting analysis of amide I band, which represents the characteristic band of the alfa-helical structure, between 1600 and 1720 cm-1 confirmed, finally, the structural stability of BSA loaded into SLMs produced by SC. In the case of SLMs prepared by SFD, this spectroscopic technique highlighted BSA structural changes which were not revealed by the other two techniques. The modifications of the secondary structure of the BSA seemed to be due to the greater number of damaging events that affected the protein, such as the denaturant action of the solvent and the aggregation phenomenon at the solid-vapour interface during freeze-drying. In conclusion, the results showed that the Spray Freeze Drying caused a structural alteration of the protein, while the Spray Congealing technology allowed to produce microparticles without denaturing the BSA secondary structure.

BSA-loaded solid lipid microparticles prepared by spray congealing and spray freeze drying.

DI SABATINO, MARCELLO;ALBERTINI, BEATRICE;PASSERINI, NADIA;RODRIGUEZ, LORENZO
2010

Abstract

The aim of this research was to explore the possibility of using two different production technologies, such as Spray Congealing (SC) and Spray Freeze Drying (SFD) in the preparation of Solid Lipid Microparticles (SLMs) containing proteins. Bovine Serum Albumin (BSA) was employed as model protein due to its well-known physical-chemical state characterization. SC technology is based on spraying a fluid consisting of a solution or suspension of active pharmaceutical ingredient in a molten carrier, which melts at a relatively low temperature (50-80°C). Conversely, in SFD process, a solution or a biphasic system containing the dissolved protein is sprayed into a cryogenic liquid (-70°C) to form droplets which freeze upon the contact with the cryogenic solution and this suspension is then freeze dried to obtain a fine powder. In the first case BSA might be denaturated by heat, while in latter case, the degrading action could result from both the use of solvents and the freeze-drying process. Six batches of microparticles employing three lipid (Precirol®ATO 5, Dynasan®114 and Dynasan®118) with different melting temperatures and containing 10% and 20 % of BSA were produced by SC. On the contrary, using SFD technology , two formulations containing 9% and 16% of BSA in the presence of Dynasan®114 as lipid carrier, lecithin and poloxamer 407 as surfactants and sucrose as lyoprotector were preparedt. The results showed process yields ranging between 65% to 80% and encapsulation efficiencies above 90% and between 80% and 90% for SC and SFD formulations, respectively. SEM analysis displayed that all batches produced by SC were spherical and not aggregated with a mean volume diameter ranging between 150-300 micron. Conversely, SFD formulations showed numerous surface fractures, which appeared or as small cracks or as noticeable lacks of lipid membrane. Physical-chemical characterization using differential scanning calorimetry (DSC), UV spectrophotometry in fourth derivative mode and Raman microspectroscopy was performed. Thermal analysis showed that the protein degradation in solid state started around 80°C. UV spectrophotometry, however, highlighted that no spectral variations occurred between aqueous solution of BSA (reference) and BSA solutions obtained by hot extraction (70-75°C) of the SLMs produced by both techniques. The Raman microspectroscopy, associated with the curve fitting analysis of amide I band, which represents the characteristic band of the alfa-helical structure, between 1600 and 1720 cm-1 confirmed, finally, the structural stability of BSA loaded into SLMs produced by SC. In the case of SLMs prepared by SFD, this spectroscopic technique highlighted BSA structural changes which were not revealed by the other two techniques. The modifications of the secondary structure of the BSA seemed to be due to the greater number of damaging events that affected the protein, such as the denaturant action of the solvent and the aggregation phenomenon at the solid-vapour interface during freeze-drying. In conclusion, the results showed that the Spray Freeze Drying caused a structural alteration of the protein, while the Spray Congealing technology allowed to produce microparticles without denaturing the BSA secondary structure.
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M. Di Sabatino; B. Albertini; N. Passerini; V.L. Kett; L. Rodriguez
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/106677
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