Adami, Renata

Liparoti, Sara

Reverchon, Ernesto

ADAMI, RENATA

LIPAROTI, SARA

REVERCHON, Ernesto

Archivio della Ricerca - Università di Salerno

     1  A new supercritical assisted atomization layout for the micronization of thermolabile compounds  Renata Adamia*, Sara Liparotia, Ernesto Reverchona,b   aDepartment of Industrial Engineering. University of Salerno Via Ponte Don Melillo, 84084 Fisciano (SA), ITALY  bResearch Centre of Nanomaterials and nanoTechnology (NANOMATES), University of Salerno, ITALY  E-mail: radami@unisa.it*   ABSTRACT A novel configuration of Supercritical Assisted Atomization (SAA) technique is proposed, that can produce microparticles with spherical morphology and controlled particle size and distribution of thermolabile compounds, difficult to be processed using other techniques, included standard SAA configuration. A vacuum system has been added, that allows to operate below the atmospheric pressure; as a consequence the drying step can be performed at lower temperatures, without damaging thermolabile materials. To verify the efficiency of the new SAA process arrangement, some compounds have been tested: Poly(l-lactide) (PLLA), bovine serum albumin (BSA), Polyethylene glycol (PEG), PEG-PLA compolymers, Poly(lactic-co-glycolic acid) (PLGA), polyvynilpyrrolidone (PVP), α-tocopherol. Process parameters, such as gas to liquid ratio and precipitation temperature, were investigated to evaluate their influence on particle size and morph logy. All the compounds were successfully processed avoiding problems of coalescence, degradation and denaturation.  INTRODUCTION The most used supercritical fluid based techniques, to produce dried microparticles, are: rapid expansion of supercritical solutions (RESS) [1], supercritical antisolvent precipitation (SAS) [2] and supercritical assisted atomization (SAA) [3, 4]. However, RESS has a limited application, since proteins and many compounds show very low solubility in supercritical carbon dioxide (SC-CO2) [5]. SAS technique has been efficiently used to micronize polymers and proteins [2, 6-8]. At the traditional temperatures used in SAS process, about 40°C [9], the particles tends to form filament in the precipitation vessel. All these techniques give not very satisfactory results when thermolable compounds have to be processed. SAA process is based on the solubilization of a controlled amount of SC-CO2 in a liquid solution in which the solute, to be micronized, has been previously dissolved. The obtained solution is atomized through a nozzle, in a precipitator operating at atmospheric pressure. Microparticles are obtained by droplet evaporation usig warm nitrogen. The presence of CO2 solubilized in the liquid solution decreases the viscosity and the surface tension of the system, improving the injection step. Droplet evaporation should be fast and complete to avoid coalescence phenomena and the impingement of the droplets on the precipitator inner surface. SAA is an efficient process to produce aerosolizable compounds and polymers [10-14]; but, in the original configuration it was not possible to micronize polymers with a low glass transition temperature (<60°C) and thermolabile compounds (some vitamins, enzymes, etc.). The precipitation temperature is selected on the basis of the temperature at which the solvent evaporates t the pressure in the precipitator. If the pressure is higher than the atmospheric pressure an higher temperatur  has to be used to allow the evaporation of the solvent. Therefore, the plant has been modified adding a vacuum system downstream the precipitation vessel.  MATERIALS AND METHODS CO2 was purchased by SON (Naples) and N2 by SOL (Caserta); acetone (99.5%), ethanol (99.5%), water and chloroform (99.5%), polyvynilpyrrolidone (PVP, Mw:10000, Aldrich Chemical Co, Milan, Italy) were used as      2  received.Poly(l-lactide) (PLLA), bovine serum albumin (BSA), Polyethylene glycol (PEG), PEG-PLA compolymers, Poly(lactic-co-glycolic acid) (PLGA), polyvynilpyrrolidone (PVP), α-tocopherol were purchased by Sigma Aldrich (Milan, Italy). The morphology of the particles was observed by a field emission-scanning electron microscope (FESEM, mod. LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany).  Particle size (PS) and particle size distribution (PSD) were measured from SEM photomicrographs using the Sigma Scan Pro Software. Histograms representing the particle size distribution were fitted using Micro al Origin Software. Furthermore, theyd were measured by dynamic laser scattering (DLS) using a Nanosizer (NanoZS Malvern Instrument) equipped with a He-Ne laser operating at 5.0 mW and 633 nm, that measures the hydrodynamic diameter of the particles. Solid state analysis of the samples (XRPD=X-ray powder diffraction) was performed using an X-ray diffractometer (mod. D8 Discover, Bruker AXS) with a Cu sealed tube source.  APPARATUS CONFIGURATION The SAA modified laboratory apparatus consisted of tw  high-pressure pumps delivering the liquid soluti n and liquid CO2 to a saturator. The saturator is a high pressure vessel (25 cm3 IV) 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 ID) injection nozzle into the precipitator (3 dm3 internal volume) operating at atmospheric pressure. A controlled flow of N2 is taken from a cylinder, heated in an electric heat exchanger and sent to the precipitator to assist liquid droplet evaporation. The saturator and the precipitator are electrically heated using thin band heaters. A tainless steel filter located at the bottom of the pr cipitator allowed powder collection and the gaseous stream flow out. The plant is completed by a condenser, thatseparates the solvent from the gas stream. The new configuration of SAA plant is reported in Figure 1: the vacuum system is composed by the condenser (C) and two vacuum pumps (VP1 and VP2) in parallel connection. SAA apparatus layout and further details on the experimental procedures were published elsewhere [15, 16].  Figure 1. SAA plant in the new configuration. (H) heat exchanger; (GP) CO2 pump; (L) liquid solution feed; (LP) liquid pump; (M) saturator; (P) precipitator; (VP1) vacuum pump; (VP2) vacuum pump; (C) condenser.       3  The new configuration using vacuum pumps, allows to set lower precipitation temperatures and to perform experiments at different reduced pressures (up to 0.65 bar). The reduction of pressure allows to operate at temperatures as low as 5°C. This temperature is obtained reducing the temperature of the nitrogen flow and taking advance of the Joule-Thomson effect, produce by CO2 expansion, that largely contributes to induce the decrease of temperature. The reduction of nitrogen flow rate allows to produce an even lower pressure in the precipitator (0.5 bar) since the overall quantity of gases to be treated by the vacuum system is smaller.  RESULTS AND DISCUSSION Several compounds have been up to now micronized using SAA under reduced pressure, starting from solutions in water or in organic solvents with a low boiling point (ethanol, acetone, chloroform). The operating conditions have been selected according to the characteristics of the compound to micronize (Table 1). Many of the polymers presented in this work, when processed using the standard SAA tended to form membrane like structures on the filter [15, 17]. This phenomenon is probably due to the softening of the particles and their a partial fusion a temperature slightly lower than the fusion temperature. Other compounds were denaturated [15, 18].  Table 1. Selection of SAA tests performed using the new SAA configuration (c=solute concentration; Tp=precipitation temperature; pp=precipitation pressure, QCO2=CO2 mass flow, QLIQ=solution mass flow, Tm=saturator temperature, pm=saturator pressure, GLR=Gas to Liquid ratio CO2/solvent w/w) solute solvent c GLR Tp pp QCO2 QLIQ  QN2 Tm pm mg/mL wCO2/wliq °C bar [gr/min] [mL/min] [L/h] °C bar BSA H2O 20 1.8 80 0.80 8.79 4.88 1224 61 80 BSA H2O 20 1.8 60 0.87 8.79 4.88 1224 60 83 PVP ethanol 20 1.8 50 1.5 8.18 4.6 1380 40 102 PLLA CHCl3 20 1 30 0.65 8.06 5.48 686 79 102 PLLA CHCl3 20 1 40 0.65 8.06 5.48 686 81 105 PLLA CHCl3 20 1 30 0.50 8.41 5.72 340 79 102 PLLA CHCl3 20 1 40 0.50 8.06 5.48 340 79 103 PEG-PLA (1:8) CHCl3 20 1.8 40 0.65 9.4 3.7 686 80 87 PEG-PLA (1:4) CHCl3 20 1.8 40 0.6 9.4 3.7 686 80 87 PEG10000 acetone 20 3.5 18 0.8 9 3 686 40 67 PEG10000 acetone 20 3.5 9 0.8 9 3 686 40 67 PEG10000 acetone 20 3.5 5 0.8 9 3 686 40 67 PEG10000 acetone 30 3.5 5 0.8 9 3 686 40 67 PEG10000 acetone 40 3.5 5 0.8 9 3 686 40 67 PEG10000 acetone 60 3.5 5 0.8-1 9 3 686 40 67 PEG-αTCP (5%) acetone 30 4 20 0.8 11 3.5 686 40 75 PEG-αTCP (10%) acetone 30  20 0.82 11 3.5 686 40 67 PLGA acetone 20 3.5 20 0.85 10.20 3.7 686 40 65  When solvent were water and ethanol, the efficiency of the vacuum system was lower than in the case of chloroform and acetone: these solvent show greater cohesive forces, that probably produced interactions n the collection filter; as a consequence, the lower pressure obtainable in the precipitator using the new SAA configuration is about 0.8-1 bar. An example of particles produced using ethanol as solvent are showed in Figure 2, where a SEM photomicrograph of PVP microparticles obtained at 50°C and 1.5 bar is reported.      4   Figure 2. SEM photomicrograph of PVP microparticles obtained using ethanol at Tp=50°C and Pp=1.5bar Using chloroform and acetone, lower reduced pressur in the precipitator were reached and, as a consequence, a lower temperature during the drying of the droplets was used. PLA-PEG copolymers or PEG were successfully processed, obtaining well defined spherical particles, as shown in the SEM photomicrograph of Figure 3.   Figure 3. SEM photomicrograph of PEG microparticles obtained by SAA at Tp=5°C For all materials, the effect of the operating conditions on the PSD are similar. The particle size increased with the compound concentration in the starting solution; whereas, the increase of precipitation temperature lead to a decrease of PS. This is due to the effect of these two parameters on the density and viscosity of the solution. Indeed, the increase of solute concentration increases the density and, as a consequence, the viscosity of the injected solution; the increase of temperature during precipitation leads to a reduction of the viscosity of the sprayed system. An example of these effects is report d in Figure 4, where the PSD in terms of volumetric cumulative percentages of PEG and PLLA are reported.       5  1.0 1.5 2.0 2.5 3.0 3.5020406080100  volume (%)diameter (µm) 5°C 9°C 18°C Figure 4. PSD of PLLA powders obtained by SAA operating at different precipitation temperatures, obtained by DLS and SEM (left); PSD of PEG powders obtained by SAA operating at different precipitation temperatures, obtained by DLS (right). The calculations were performed in terms of volumetric cumulative percentages. PS also depends on the material to micronize. In particular, considering polymers processed at 20 mg/ml, PLLA particles show a mean diameter of 1-1.5 µm, PEG a me n diameter at 1.9-2.5 µm and PEG-PLA copolymers a mean diameter of 0.4-0.6 µm, the variation depending on the precipitation temperature and the reduced pr ssure in the precipitator. Using SAA in the new configuration, it is also possible to produce coprecipitates. Lipophilic vitamin α-tocopherol, was coprecipitated with PEG, with the aim to improve stability and bioavailability of the vitamin. Different percentages of vitamin (5% and 10%) were loaded in PEG and spherical particles, with a mean diameter between 1 and 2 µm (Figure 5), were obtained, with a high encapsulation efficiency.   Figure 5. PEG loaded 10% (w) of α-tocopherol microparticles obtained by SAA The XRPD analyses demonstrated that the SAA process doe  not modify the structure of the compound but often induces the formation of structures with a lower degre  of crystallinity compared to the unprocessed material, as      6  a consequence of the faster rate of precipitation cmpared to the traditional crystallization. In Figure 6 an example of XRPD is reported: it is possible to note that, in the case of PLA, the spectra of the processed copolymers have the halo characteristic of the amorphous structures.  Figure 6. XRPD of untreated PLLA and of the particles obtained by SAA process CONCLUSION AND PERSPECTIVES The new plant configuration for SAA processing, with the implementation of the vacuum system is effectiv  in the reduction of the precipitation pressure and, correspondingly, it is possible to operate at lower temperatures. The process is more efficient when chloroform and acetone are used, with respect to water and ethanol. Several compounds with biological activity with a low glass transition and denaturation temperature hav  been successfully processed avoiding problems of coalescence, degradation and denaturation.  Up to now has been possible to perform SAA operation below the atmospheric pressure and at temperatures as low as 5°C, in future a cooling system could be added to the precipitation vessel to operate at temperature below zero and process materials as labile as vaccines.  REFERENCES [1] MATSUYAMA, K., MISHIMA, K., HAYASHI, K.I., ISHI KAWA, H., MATSUYAMA, H., HARADA, T., Journal of Applied Polymer Science.Vol. 89(3), (2003), p. 742. [2] BAHRAMI, M., RANJBARIAN, S., The Journal of Supercritical Fluids.Vol. 40(2), (2007), p. 263. [3] REVERCHON, E., Industrial and Engineering Chemistry Research.Vol. 41(10), (2002), p. 2405. 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A new supercritical assisted atomization layout for the micronization of thermolabile compounds

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