ADAMI, RENATA

LIPAROTI, SARA

REVERCHON, Ernesto

English

Archivio della Ricerca - Università di Salerno

Ninth Conference on Supercritical Fluids and Their Applications Sorrento, Italy, 05-08 September 2010 – Ed. E. Reverchon  223 Production of microspheres of Bovine Serum Albumin by Supercritical Assisted Atomization at reduced pressure S. Liparoti*, R. Adami, E. Reverchon Dipartimento di Ingegneria Chimica e Alimentare, Università degli Studi di Salerno. Via Ponte Don Melillo, 84084 Fisciano (SA), Italy.  * sliparoti@unisa.it; fax: +39-089964057  Supercritical Assisted Atomization (SAA) is a very efficient technique to micronize water soluble compounds. However many of these compounds, such as bioactive molecules, proteins and polymers, are sensitive to relatively high temperatures and are difficult to be processed using traditional micronization techniques. Even the relatively low precipitation temperatures, used during SAA, can compromise their stability. Therefore, in this work, SAA was modified adding a vacuum system downstream the precipitation vessel to reduce the precipitation pressure and allow to operate at temperatures that do not degrade thermolabile compounds. This new SAA arrangement has been used to obtain microparticles of Bovine Serum Albumin (BSA) with particle size distributions suitable for aerosol delivery. The particles produced have a mean diameter ranging between 0.2 and 4 μm and are amorphous at all the investigated temperatures. FTIR tests showed that BSA structure is preserved. INTRODUCTION Albumin is the most abundant serum protein. It is a globular protein with the approximate shape of a prolate spheroid [1]. It has several pharmacological and physiological functions:   carrier of metals, fatty acids, cholesterol, bile pigments and drugs;  regulator of the osmotic pressure;  distributor of fluids between different compartments. In general, albumin is the major antioxidant in the human plasma; many antioxidant activities of albumin result from its ligand-binding capacity. The flexibility of the albumin structure allows the adaptation of the protein sites to ligands; moreover, its tridimensional structure provides many binding sites. Particularly the cationic ligands, like copper and iron, and transition metals, that can generate free radicals very quickly in presence of oxygen, do not form free radicals when are bounded with BSA.  The pulmonary route is a not invasive route for administering proteins for systemic therapy. For an effective inhalation fine particles ranging between 1 and 5 μm are needed. The most used processes to obtain aerosolizable powders are spray drying, spray freeze-drying, lyophilization and emulsion drying. These processes have some limits: they can denature the proteins and traces of the solvents remain inside the particles.  BSA is very sensitive to high temperatures. The process of denaturation starts at about 65°C, at this temperature the denaturation is reversible; upon 65°C the unfolding of the BSA protein chain is irreversible [2, 3, 4, 5]. Spray drying was proposed as micronization technique for BSA [6]. The temperature used during the process induced several changes in the protein structure. Another technique to micronize BSA is spray drying under reduced pressure [7]; however the particles obtained have a mean diameter ranging from 15 to 24 μm.  Liparoti et al. 224 Supercritical assisted atomization (SAA) is a micronization technique [8] in which supercritical CO2 acts as the atomizing medium. Controlled quantities of SC-CO2 are solubilized in the liquid solution formed by the solvent and the solute. The solution is, then, sent to an injector and sprayed into a near atmospheric precipitator. The reduced viscosity and surface tension of the solution and the fast release of SC-CO2 contribute to a very efficient atomization. Two atomization processes take place: the pneumatic atomization, in which primary droplets are produced, and the secondary atomization, in which secondary droplets are formed from the break of the primary droplets.  Until now SAA has been used to micronize enzymes, as lysozyme [9]; but, has never been used to micronize proteins that are more sensitive to high temperatures.  In this work we propose a modified SAA scheme in which precipitation is obtained under reduced pressure and BSA processing to demonstrate the feasibility of the process.  MATERIALS AND METHODS Materials Bovine Serum Albumin (BSA) was supplied from Sigma Aldrich (66 kDa, V fraction, γ globulin free). Distilled water was produced at DICA. Carbon dioxide (CO2) (purity 99%) was purchased from SON (Napoli, Italy); nitrogen (N2) (purity 99%) was purchased from SOL (Marcianise, Italy). Apparatus The SAA plant is schematically reported in Figure 1. It consists of two high-pressure pumps delivering the liquid solution and liquid CO2 to the saturator. The saturator is a high pressure vessel (I.V. 25 cm3) loaded with stainless steel perforated saddles which assure a large contact surface between liquid solution and CO2. The solution obtained in the saturator is sprayed through a thin wall (80 µm diameter) injection nozzle into the precipitator (I.V. 3 dm3) operating at atmospheric pressure. A controlled heated flow of N2 is sent to the precipitator to assist liquid droplets evaporation. A stainless steel filter, located at the bottom of the precipitator, allows the powder collection and the gaseous stream flow out. Downstream the precipitator, a condenser separates the liquid stream from the inert gas and a vacuum pump allows to reduce the pressure in the precipitator.  Ninth Conference on Supercritical Fluids and Their Applications Sorrento, Italy, 05-08 September 2010 – Ed. E. Reverchon  225 CO2 PUMPLIQUID PUMPLCVENTN HPMVACUUMPUMP  Figure 1 Schematic representation of the SAA apparatus: (C) CO2 cylinder; (L) liquid solution; (N) N2 cylinder; (H) heat exchanger; (M) saturator; (P) precipitator; (S) condenser. Analytical Methods Particles produced by SAA were observed by a Scanning Electron Microscope (SEM) coupled with a field emission source.  Particle size (PS) and particle size distribution (PSD) were measured from SEM photomicrographs using the Sigma Scan Pro Software. Fourier transform infrared (FT-IR) spectra were obtained via M2000 FTIR, at a resolution of 0.5 cm-1. All spectra were smoothed using a 13-point Savitzky–Golay smoothing function to remove white noise. Composition bands of the amide I (1600–1700 cm-1) and amide III (1200-1300 cm-1) were determined using the inverted second derivative [10]. RESULTS AND DISCUSSION SAA process is based on the solubilization of a controlled amount of CO2 in the liquid solution. When water is used as liquid solvent, a limited amount of CO2 is solubilized in the liquid, at the ordinary SAA pressures and temperatures in the saturator. CO2 not solubilized enhances the pneumatic effect in the injector, supplementing the nozzle pressure difference and improving the primary atomization. The SAA operating conditions used in this work were preliminary selected, on the basis of previous experiences on this process [11, 12]. Saturator conditions were set at 85°C and 105–110 bar; CO2 flow rate ranged between 9 and 10 g/min, solution flow rate ranged between 5 and 6 mL/min. Using these process conditions the residence time in the saturator (5–6 min) is long enough to allow an efficient contact between CO2 and liquid mixture. After the selection of the saturator parameters the effect of pressure and temperature at the precipitator was investigated. The high pressure drop, caused by the presence of the porous filter and by the presence of the deposited particles, increases the precipitator pressure. Therefore, a vacuum system is used to reduce the pressure in the precipitator. The partially reduction of the pressure allows to set lower precipitation temperatures to process thermolabile compounds. Keeping constant the operative conditions in the saturator and the temperature in the precipitation vessel we have investigated the effect of the precipitation pressure. An example Liparoti et al. 226 of preliminary experiments is shown in Figure 2. The particles have a spherical morphology but the ones obtained at a lower pressure are collapsed. This is because the structure of the particles is influenced by the evaporation rate of the solvent. When we set a lower precipitation pressure the vapor pressure of the solvent decreases so the evaporation starts at lower temperature. If the pressure in the precipitator decreases but the temperature remains the same (as in the tests showed in Figure 2) the evaporation rate increases and the structure of the final particles is not stable. These results suggest that to obtain particles with a regular morphology it is necessary to reduce not only the pressure, but also the temperature in the precipitation vessel.   Figure 2. (a) BSA microparticles obtained by SAA operating at 2.4 bar (a) and 1.6 bar (b) in the precipitator Therefore, some tests were performed changing the temperature in the precipitation vessel and fixing precipitation pressure. Figure 3 shows the SEM images related to these tests. As expected the particles obtained at lower temperatures show a more regular morphology.            Figure 3 (a) BSA microparticles obtained by SAA using a precipitation temperature of 60°C (a) or 100°C (b). The mean diameter of the particles is about 1 µm in all the cases. The cumulative particles size distribution (PSD) (Figure 4) shows that the temperature have a minor influence the on dimension of BSA particles. Ninth Conference on Supercritical Fluids and Their Applications Sorrento, Italy, 05-08 September 2010 – Ed. E. Reverchon  227 0 1 2 3020406080100 Tcam=60°C QN2=1750 L/h Tcam=80°C QN2=1750 L/h Tcam=100°C QN2=1750 L/hvolume, %diameter, m Figure 4 Cumulative PSD relating to the tests at different precipitation temperature. As we have previously discussed the protein chain of BSA unfolds when temperature rises above 65°C. To verify that the protein chain does not unfold during the SAA with the new configuration FTIR analysis are performed. Figure 5 shows FTIR spectra of the untreated BSA and of the micronized BSA. The investigated bands are amide I and amide III. 4000 3000 2000 1000adsorbance (arb.)wavelength BSA untreated60°C80°C100°C   Figure 5 FTIR spectra of untreated and micronized BSA. The three main regions of the amide I band are: α-helix around 1653–1658 cm-1, β-sheet around 1630–1641 cm-1 and molecular turn of β-sheet around 1660–1688 cm-1. In the region of amide III there are α-helix band around 1284–1290 cm-1 and the β-sheet band around 1230–1242 cm-1. The relative intensity of the peaks related to the bands can be used for comparison purposes. It shows that at 100°C the area related to the peaks of α-helix increases and the area related to the peaks of β-sheet decreases whereas the analysis of the spectra related to the particles obtained at 60°C does not show any reduction. Liparoti et al. 228 CONCLUSIONS SAA process at reduced pressure has been successfully implemented. BSA microparticles, obtained by SAA, are spherical and have a mean diameter suitable for aerosolizable formulations. As demonstrated by FTIR analysis, the structure of the protein was preserved during the process using the new configuration.  REFERENCES [1] McClellan S. J., Franses E. I. (2003) Effect of concentration and denaturation on adsorption and surface tension of bovine serum albumin. Colloids and Surfaces B: Biointerfaces 28 63/75.  [2] Kuznetsow, A. N., Ebert, B., Lassmann, G. and Shapiro, A. B. (1975). Adsorption of small molecules to Bovine serum Albumin studied by the Spin-Probe method. Biochim. Biophys. Acta379; 139-146. [3] Lin V. J. C. and Koenig J. L. (1976). Raman studies of bovine serum albumin. Biopolymers15; 203-218. [4] Oakes J. (1976). Thermally Denatured Proteins. J. Chem. Soc. Faraday I72; 228-237 [5] Ruegg M., Moor U., and Blanc B. (1977). A calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate. J. Dairy Res. 44; 509-520 [6] Costantino H.R., Firouzabadian L., Hogeland K., Wu C., Beganski C., Carrasquillo K.G., Còrdova M., Griebenow K., Zale S.E., Tracy M.A. (2000). Protein Spray-Freeze Drying. Effect of Atomization Conditions on Particle, Size and Stability, Pharmaceutical Research, Vol. 17, No. 11. [7] Freitas S., Merkle H.P., Gander B. (2004). Ultrasonic atomisation into reduced pressure atmosphere—envisaging aseptic spray-drying for microencapsulation. Journal of Controlled Release 95 (2004) 185– 195 [8] Reverchon E. (2002). Supercritical-Assisted Atomization To Produce Micro- and/or Nanoparticles of Controlled Size and Distribution, Ind. Eng. Chem. Res. 2002, 41, 2405-2411. [9] Adami R., Sesti Osseo L., Reverchon E. (2009), Micronization of Lysozyme by Supercritical Assisted Atomization. Biotechnology and Bioengineering, Vol. 104, No. 6, December 15. [10] Fu K., Griebenow K., Hsieh L., Klibanov A.M., Langer R. (1999) FTIR characterization of the secondary structure of proteins encapsulated within PLGA microspheres. Journal of controlled release Roche M., Rondeau P., Singh N.R.,Tarnus E., Bourdon E. (2008) The antioxidant properties of serum albumin., FEBS Letters 582 (2008) 1783–1787. [11] Della Porta G., De Vittori C., Reverchon E. (2005) Supercritical Assisted Atomization: a novel technology for microparticles preparation of an asthma controlling drug. AAPS PharmSciTech E421-E428. [12] Reverchon E. and Della Porta G. (2003). Micronization of antibiotics by supercritical assisted atomization. J. Supercrit. Fluids 26, 243-252. 

Production of microspheres of Bovine Serum Albumin by supercritical assisted atomization at reduced pressure

https://www.iris.unisa.it/bitstream/11386/3016398/1/2010%20Sorrento%20M16.pdf

Production of microspheres of Bovine Serum Albumin by supercritical assisted atomization at reduced pressure

Abstract

Similar works

Full text

Available Versions

Archivio della Ricerca - Università di Salerno