Nanosizing of sodium ibuprofen by SAS method

Nanosizing of sodium ibuprofen by SAS method

Powder Technology 270 (2015) 378–386 Contents lists available at ScienceDirect Powder Technology journal homepage: www.elsevier.com/locate/powtec N...

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Powder Technology 270 (2015) 378–386

Contents lists available at ScienceDirect

Powder Technology journal homepage: www.elsevier.com/locate/powtec

Nanosizing of sodium ibuprofen by SAS method Natália Mezzomo a,1, Sibele R. Rosso Comim a,1, Carlos E.M. Campos b, Sandra R.S. Ferreira a,⁎ a b

EQA-CTC/UFSC, Chemical and Food Engineering Department, Federal University of Santa Catarina, C.P. 476, CEP 88040-900 Florianópolis, SC, Brazil Department of Physics, Federal University of Santa Catarina, 88040-900 Florianópolis, Brazil

a r t i c l e

i n f o

Article history: Received 21 July 2014 Received in revised form 1 October 2014 Accepted 24 October 2014 Available online 30 October 2014 Keywords: Flexibility Nanosize SAS process Crystallinity Phase equilibria

a b s t r a c t The micro/nanosizing of drug particles has been identified as a potentially effective and broadly applicable approach. Sodium ibuprofen is a chiral nonsteroidal anti-inflammatory drug. This work aimed to recrystallize particles of sodium ibuprofen, reducing its particle size, using a Supercritical Anti-solvent (SAS) technique performed in a modified supercritical fluid extraction unit. For this purpose, the phase equilibrium of the system sodium ibuprofen + acetone + CO2 was also investigated at temperatures ranging from 35 to 55 °C. Phase equilibrium data exhibited solid–vapor–liquid and vapor–liquid transitions. The average particle sizes of the SAS-precipitated ibuprofen were below 380 ± 84 nm for all the SAS conditions tested, reducing the original dimension of the samples from micrometric to nanometric order. The best SAS operational conditions, in order to produce the lowest estimated particle size and higher crystallinity were, respectively, using 0.5 and 1.0 mgibuprofen/mL, and 1 mLsolution/min, 1 kgCO2/h, 110 bar and 35 °C. The DSC results indicated that, besides reducing the ibuprofen particle size, the SAS process appears to have changed the original sodium ibuprofen to the acid form. The PXRD and RAMAN results indicated that the SAS process at 1 mgibuprofen/mL, 1 mLsolution/min, 1 kgCO2/h, 110 bar and 35 °C is the best condition to obtain ibuprofen particles with higher crystallinity. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) used to reduce fever and to treat pain or inflammation. It is presented in many formulations (powders, capsules, tablets, etc.), and it is available over the counter in most countries as one of the core medicines listed in World Health Organization's “Essential Drugs List” [1]. The nanosizing of drug particles has been identified as a potentially effective and broadly applicable approach. For example, smallerdiameter particles have a faster dissolution rate, with potentially higher activity and easier absorption. Other distinct advantages include tissue or cell-specific targeting of drugs, easier dispersion throughout the body, higher stability against enzymatic degradation, and the reduction of unwanted side effects [2–5]. Disadvantages related to nanoscale drug particles are the difficulty in their production, and also their physical instability, which may lead to particle aggregation, causing problems related to drug storage and administration [3,5]. Traditional techniques for particle size reduction such as mechanical milling and precipitation– condensation present considerable success in the nanosizing of drug particles, but concerns including the broad particle size distribution and the excessive use of organic solvent remain to be addressed [6].

⁎ Corresponding author. Tel.: +55 48 37219448. E-mail address: [email protected] (S.R.S. Ferreira). 1 Tel.: +55 48 37219448.

http://dx.doi.org/10.1016/j.powtec.2014.10.036 0032-5910/© 2014 Elsevier B.V. All rights reserved.

In the past decade, supercritical fluid techniques have gained significant attention in many fields, such as extraction, chromatography, chemical reaction engineering, organic and inorganic synthesis, waste management, material processing, porous materials, and material production for pharmaceutical applications [7–12]. Supercritical fluids present low viscosity, permitting matrix penetration as gas-like characteristic; liquid-like density, promoting solute solubilization; high diffusion; and near-zero surface tension. At the critical point, the density of the gas phase becomes equal to that of the liquid phase, and the interface between gas and liquid disappears. Supercritical CO2 (scCO2) is the most widely used supercritical fluid due to its relatively low critical conditions (Tc = 31.1 °C, Pc = 7.38 MPa), nontoxicity, nonflammability, and low price [13]. Particle processing is one of the major developments of supercritical fluid applications in industrial fields such as the chemistry, pharmaceutical, cosmetic, and agriculture and food industries because, besides the novelty related to process characteristics, it also accommodates the principles of green chemistry [12]. Various modified supercritical techniques based on different nucleation and growth mechanisms of precipitating particles have been developed [14]. The well-known techniques for particle formation using scCO2 include the rapid expansion of supercritical solutions (RESS) [15] and a variety of antisolvent processes such as Gas Antisolvent (GAS) [16], Aerosol Solvent Extraction Systems (ASES) [17], Particles from Gas-saturated Solutions (PGSS) [35], Supercritical Antisolvent (SAS) processes [18–20], and Solution-enhanced Dispersion by Supercritical fluids (SEDS) [21]. In the SAS process, the scCO2 and the liquid solution are simultaneously introduced into the high-pressure vessel using or not a specially

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designed coaxial nozzle. When the solution droplets reach the scCO2, a rapid mutual diffusion at the interface of the droplets and the scCO2 instantaneously takes place, inducing phase separation and supersaturation of the solute in scCO2, leading to its nucleation and precipitation [14]. The supercritical fluid is used both as anti-solvent for its chemical properties and as a ‘spray enhancer’ by mechanical effects. The temperature and pressure, together with accurate metering of flow rates of solution and supercritical fluid, provide uniform conditions for particle formation. Morphology and particle size of the product (formed particles) can be adjusted by means of the process parameter optimization [13,22]. Considering the cited literature information, this work aimed to recrystallize sodium ibuprofen particles by means of the Supercritical Anti-solvent (SAS) technique performed in a modified/adapted supercritical fluid extraction unit. For this purpose, the phase equilibrium of the system sodium ibuprofen + acetone + CO2 was also investigated in order to suggest adequate conditions for the precipitation SAS assays. Therefore, the present study also seeks to evaluate the adaptation of the extraction unit to perform a SAS process, by studying the process parameters on the recrystallized particles of sodium ibuprofen. 2. Materials and methods 2.1. Materials Sodium ibuprofen (Sigma Aldrich, Brazil) was used as the solute to perform the precipitation processes and phase equilibrium assays. In order to prepare the precipitation solution, different concentrations of sodium ibuprofen were solubilized by the primary solvent acetone (P.A., Nuclear, CAQ Ind. e Com. LTDA., Brazil), using constant agitation and heat application (40 °C, 10 min) until a complete solute solubilization

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was reached. The processes used 99.9% pure carbon dioxide (White Martins, Brazil), delivered at 60 bar.

2.2. Conformation of SFE equipment to SAS process A supercritical fluid extraction (SFE) unit previously detailed by Zetzl et al. [23], and presently shown in Fig. 1, was adapted to perform a Supercritical Anti-solvent (SAS) process. Both techniques (SFE and SAS) require the use of a pump (M111, Maximator, Germany), used to provide the solvent or the antisolvent (CO2) at the desired high pressure conditions. In order to warrant the correct functioning of the pump, CO2 must be supplied in liquid form, which requires the use of a heat exchanger (cooler — C10-K10, TermoHaake) (1 from Fig. 1) in order to condense the gaseous fluid from the CO2 bottle outlet. The oscillation frequency of the piston is controlled by the throttle valve (VT from Fig. 1). The gear ratio of the booster piston is 1:130. The system pressure is controlled by the spring-loaded backpressure regulator (Tescom Cat. no 26-1761-24-161 — V1 from Fig. 1). The piston sensor opens when the required pressure is reached. Subsequently, the preheated fluid flows from the valve box (4 from Fig. 1) back to the sucking section inside the condenser (1 from Fig. 1). Consequently, the pump continuously produces a compressed CO2 flow in a closed loop. The closed loop assembly allows constant solvent supply with low-pressure fluctuation. Before and behind the valves, the expansion-induced freezing of the CO2 flow (Joule Thompson effect) may lead to a complete blocking of the tubes by dry ice particles. Therefore, all relevant valves were placed in a tempered heating bath (4 from Fig. 1). Changing the original configuration, the extraction/precipitation chamber was heated in a second tempered heating bath, regulated at the operational temperature desired.

Fig. 1. Flow sheet of the supercritical fluid extraction [23].

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Fig. 2 presents the modifications of the extraction unit (Fig. 1) with the purpose of performing the precipitation/encapsulation processes using scCO2. The precipitator cell used to perform the SAS process was the same extraction chamber from the SFE unit, assembled in AISI 316 stain-less steel with vessel dimensions: volume of 103.28 mL, height of 31.6 cm and inner diameter of 2.012 cm (6 from Fig. 2). The vessel was thermostatically controlled by a regulated water bath (4 from Fig. 2 — DC30-B30, Thermo Haake). One porous frit, screen size 1 μm, was placed at the bottom of the precipitator chamber and used to collect the precipitated particles. An air driven piston pump (3 from Fig. 2 — M111, Maximator) and an HPLC pump (4 from Fig. 2 — Constametric 3200 P/F, Thermo Separation Processes) were used to feed the scCO2 and the organic solution (organic solvent + sodium ibuprofen) into the vessel. The two streams (CO2 and solution) were mixed by means of a concentric tube nozzle placed at the top of the precipitation vessel. The liquid organic solvent is solubilized by the CO2 and, through system depressurization, the organic solvent was deposited in a glass flask (8 from Fig. 2) and the flow rate of gaseous CO2 was measured by a rotameter (RM from Fig. 2 — 10A61, ABB Automatic Products). Temperature and pressure conditions were measured with instruments directly connected to the precipitation vessel, with accuracies of ±0.5 °C and ±2 bar, respectively.

Phase transitions were visually observed by pressure manipulation using the syringe pump. In the experimental procedure, a precise amount of the sodium ibuprofen solution dissolved in acetone (1 mg/mL) was weighed in an analytical balance (Ohaus, Model AS200S, NJ, USA) with ±0.0001 g of precision and loaded into the equilibrium cell. A known amount of carbon dioxide at 5 °C and 100 bar was loaded into the equilibrium cell using the syringe pump, resulting in an accuracy of ±0.005 g in CO2 loadings until a desired mass fraction was achieved (from 60 to 95% of CO2). The quantity of carbon dioxide loaded is accounted by volume dislocation in the syringe pump. Therefore, CO2 density, at the fixed conditions of temperature and pressure, obtained from the NIST Chemistry Webbook [36], was used for the volume– mass conversion. The cell content was kept at continuous agitation with a magnetic stirrer and a Teflon-coated stirring bar. At the desired temperature, the pressure system was increased up to the formation of a one-phase system. At this point, the pressure was slowly decreased (at a rate of 4 bar/min) until incipient formation of a new phase. This procedure was repeated at least two times for each temperature (35 to 55 °C) and global composition (sodium ibuprofen/acetone solution and CO2) tested.

2.3. Phase equilibrium apparatus and experimental procedure

2.4. Precipitation processes of sodium ibuprofen

Phase equilibrium experiments were performed using the static method. The experimental apparatus and the procedure are well described in a variety of studies [24–27], based on the work of Oliveira et al. [28]. The main components of the experimental apparatus were an equilibrium cell, with a maximum internal volume of 27 mL and two sapphire windows (for light entrance and for visual observation), an absolute pressure transducer (Model 511, Huba Control, Würenlos/ Denmark), a thermocouple and a syringe pump (260HP Teledyne Isco, Lincoln/NE/EUA) with pressure range from 0.7 to 655.2 ± 0.5 bar.

Precipitation process of the sodium ibuprofen was performed in the SAS unit detailed in Subsection 2.2. The effect of the precipitation conditions of pressure (80, 110 and 140 bar), temperature (35, 45 and 55 °C), solution flow rate (1.0, 2.0 and 3.0 mL/min) and extract concentration on the feed solution (0.5, 1.0 and 1.5 mg/mL), at a constant CO2 flow rate of 1 kgCO2/h, was evaluated in respect to the particle characteristics (size, morphology and crystallinity), totalizing 11 assays (one condition in triplicate to test the technique replication). The primary solvent used in feed solution was acetone P.A. due to its high solvency power of the

Fig. 2. Flow sheet of the Supercritical Anti-solvents (SAS) unit adapted from the equipment from Zetzl et al. [23].

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sodium ibuprofen at atmospherical conditions and its high solubility in supercritical CO2 at the precipitation conditions. This procedure is described in detail by Mezzomo et al. [29]. Briefly, the experiments started by pumping pure CO2 into the precipitator vessel. When the desired operating conditions (temperature, pressure and CO2 flow rate) were achieved and remained stable, the solution was fed into the precipitator. After the injection of a pre-defined amount of solution (approximately 30 mL), the liquid pump was stopped and only pure CO2 was pumped inside the cell for 15 min in order to guarantee total drying of the particles. Subsequent to the decompression, the precipitated particles retained in the frit were collected for the particle analysis described in Subsection 2.4. All the samples were stored at temperatures below −10 °C and protected from light to avoid decomposition of the product. A conventional crystallization technique, lyophilization, was also used to provide drug particles, for comparison with the particles formed by the SAS method. Therefore, a lyophilizer (LD 1500, Terroni) was used with a frozen aqueous solution of sodium ibuprofen (50 mg/mL) for 72 h until complete drying of the particles was reached. 2.5. Particle analysis 2.5.1. Scanning electron microscopy (SEM) Samples of the powder collected from the precipitators (SAS unit and lyophilizer) were analyzed by a scanning electron microscope (SEM) model JEOL JSM-63990LV. A gold sputter was used to cover the samples with a thin layer of gold to allow light reflection for particle evaluation. An estimation of the mean particle size was measured by ZEISS image analysis software. Micrography was performed in quadruplicate for each sample and the estimated mean particle size was measured at least five times for each micrograph. The procedure is described by Mezzomo et al. [30]. 2.5.2. Differential scanning calorimetry (DSC) analysis Thermal analyses of the precipitated samples were performed with a Mettler TA 4000 differential scanning calorimeter. Samples were analyzed under nitrogen atmosphere in temperatures between − 10 and 120 °C with a heating rate of 5 °C/min. Some samples (original sodium ibuprofen, lyophilized sample and two SAS samples, selected randomly) are analyzed until 250 °C. DSC analyses were conducted in order to estimate modifications of the composition, degree of crystallinity and melting temperature of the particles caused by the SAS process. This procedure is also described in detail by Mezzomo et al. [30].

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Fig. 3. P–w phase equilibrium data for the ternary system (sodium ibuprofen + acetone + CO2 from 35 °C to 55 °C), SVLE: solid–vapor–liquid equilibrium; VLE: vapor–liquid equilibrium; wCO2: mass fraction of carbon dioxide (CO2), in comparison with literature data [31]. *SAS conditions.

were performed over 2θ angles ranging from 3 to 90° with a scan step size of 0.016° and a scan step time of about 10 s. All measurements were performed in triplicate at room temperature.

2.5.4. RAMAN Spectroscopy Raman spectra were collected in backscattering geometry using an L4× objective coupled to a PeakSeeker 785 (RAM-PRO-785) Raman system operating with a diode laser of 785 nm and 150 mW at the source. Collected Raman radiation was dispersed with a grating and focused on a Peltier-cooled charge-coupled device CCD detector. All spectra were recorded in the spectral window 200–1800 cm−1 with the same acquisition time (30 s) at room temperature.

2.6. Statistical analysis Results were statistically evaluated by a one-way analysis of variance (ANOVA), applied using the Software Statistica for Windows 6.0 (Statsoft Inc., USA) in order to detect significant differences among values. The significant differences at the level of 5% (p b 0.05) were analyzed by the Tukey test.

2.5.3. Powder X-ray Diffractometry (PXRD) The particles were also characterized using an X-ray Diffractometer (X'pert PRO Multi-purpose, PANalytical), where the X-ray source was Cu Kα radiation (λ = 1.5418 Å), powered at 45 kV and 40 mA. Scans Table 1 Phase equilibrium experimental data for the system sodium ibuprofen + acetone + CO2. σ (bar)

Transition type

wCO2 = 0.810 35 61.9 45 70.5 55 83.2

0.5 0.1 0.3

SVLE-BP SVLE-BP VLE-BP

wCO2 = 0.914 35 69.0 45 81.0 55 92.0

0.5 0.2 0.2

SVLE-BP SVLE-BP VLE-BP

wCO2 = 0.955 35 76.6

0.2

SVLE-BP

T (°C)

P (bar)

σ (bar)

Transition type

wCO2 = 0.877 35 65.0 45 77.7 55 90.0

0.1 0.6 0.6

SVLE-BP SVLE-BP VLE-BP

wCO2 = 0.934 35 73.2 45 82.8

0.6 0.4

SVLE-BP SVLE-BP

T (°C)

P (bar)

T: Temperature (°C); P: Pressure (bar); σ: standard deviation (bar); SVLE: solid–vapor– liquid equilibrium; VLE: vapor–liquid equilibrium; BP: bubble point; wCO2: mass fraction of carbon dioxide (CO2).

Fig. 4. Solid–liquid phase equilibrium for the system CO2 + ibuprofen for wCO2 = 0.877 at 35 °C.

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3. Results and discussion 3.1. Phase equilibrium data Phase equilibrium data, obtained for the ternary system sodium ibuprofen + acetone + CO2, are presented in Table 1 for temperature range from 35 °C to 55 °C and CO2 mass content (w) from 81.0% to 95.5%. The table shows the equilibrium results in terms of transition pressure and presents the associated experimental error for each condition represented by the standard deviation of the replicate measurements (σ). The pressure–composition (P–w) diagram for the results showed in Table 1 is presented in Fig. 3, while Fig. 4 presents the solid–liquid equilibrium for the system with a carbon dioxide content of 87.7% at 35 °C. As can be observed in Table 1 and from the pressure–composition diagram (Fig. 3), experimental measurements revealed the occurrence of vapor–liquid (VLE), and solid–vapor–liquid (SVLE) transitions with

bubble points. Due to the low amount of ibuprofen for the CO2 mass fraction of 0.955 (35 °C) the system seemed to have only suspended particles. No precipitation at the bottom was observed. Phase equilibrium data presented by Chiu et al. [31] for the system CO2 + acetone at 35 °C (Fig. 3) shows that the addition of sodium ibuprofen at the studied concentration appears not to affect the vapor–liquid transitions of the antisolvent + solvent system. Solid particles were observed on the liquid phase at temperatures of 35 °C and 45 °C, possibly due to low temperature and the proximity of organic solution saturation. As explained in Subsection 2.1, at 40 °C sodium ibuprofen is completely soluble in acetone and the addition of CO2 to the sodium ibuprofen +acetone solution causes the precipitation of ibuprofen particles at temperatures of 35 °C and 45 °C (Table 1), with a small quantity precipitated at 45 °C. Therefore, it can be inferred that CO2 acts as an anti-solvent in the system and that sodium ibuprofen solubility increases with temperature increase. For CO2 mass content above 93.4% at 45 °C and above 91.4% at 55 °C no phase separation was observed. However, changes of appearance of

Fig. 5. Electronic micrographs of ibuprofen processed by Supercritical Anti-solvent (SAS) process, lyophilization and of original sodium ibuprofen (non-processed). Assays: (1): 1 mg ibuprofen/mL, 1 mL solution/min, 1 kgCO2/h, 80 bar and 35 °C; (2):1 mg ibuprofen/mL, 1 mL solution/min, 1kgCO2/h, 110 bar and 35 °C; (3): 1 mg ibuprofen/mL, 1 mL solution/min, 1 kgCO2/h, 140 bar and 35 °C; (4):1 mg ibuprofen/mL, 1 mL solution/min, 1 kgCO2/h, 110 bar and 45 °C; (5): 1 mg ibuprofen/mL, 1 mL solution/min, 1 kgCO2/h,110 bar and 55 °C; (6): 0.5 mg ibuprofen/mL, 1 mL solution/min, 1 kgCO2/h, 110 bar and 35 °C; (7): 1.5 mg ibuprofen/mL,1mL solution/min, 1 kgCO2/h, 110 bar and 35 °C; (8): 1 mg ibuprofen/mL, 2 mL solution/min, 1 kgCO2/h, 110 bar and 35 °C; (9): 1 mg ibuprofen/mL, 3 mL solution/min, 1 kgCO2/h, 110 bar and 35 °C; (10) Lyophilized sodium ibuprofen;(11) Commercial sodium ibuprofen (original without processing).

N. Mezzomo et al. / Powder Technology 270 (2015) 378–386 Table 2 Operational conditions and estimated particle diameters of original sodium ibuprofen and ibuprofen processed by Supercritical Anti-solvent (SAS) process. CO2 flow Solution Assay Ibuprofen Pressure Temperature Estimated concentration flow rate rate (kg/h) (bar) (°C) particle (mL/min) (mg/mL) diameter (nm)1 1 1 1 1 2(a) 1 1 1 2(b) 2(c) 3 1 1 1 4 1 1 1 5 1 1 1 6 0.5 1 1 7 1.5 1 1 8 1 2 1 9 1 3 1 Lyophilized sodium ibuprofen Original sodium ibuprofen (non-processed)

240 ± 87a 210 ± 17a

80 110

35 35

140 110 110 110 110 110 110

35 264 ± 80a 45 127 ± 9b 55 153 ± 47b 35 87 ± 28c 35 209 ± 15a 35 207 ± 47a 35 –2 126 ± 25 (×103)α 144 ± 87 (×103)α

1

Same letter in same column indicates no statistical difference between values. Not presented in the table because the presence of big filaments is not accounted in the measurement (particle diameter without considering big filaments = 156 ± 67). 2

the single phase, from transparent to cloudy and then from transparent again, were observed. These observations might be related to the change of system from supercritical fluid to vapor phase. 3.2. Morphology and size of ibuprofen particles The SAS method produced ibuprofen particles in all precipitation conditions tested. Fig. 5 shows the electronic micrographs of the particles obtained by different SAS operational conditions, together with the lyophilized sample and the original sodium ibuprofen (nonprocessed sample). According to Fig. 5, the SAS process reduced the original size of sodium ibuprofen particles (Fig. 5: 1–9, compared to 11), from micrometric to nanometric order, as can be clearly observed by the size indications in the micrographs. In the same way, the results for the lyophilization process were statistically similar (micrometric size) (Fig. 5: 10) to the original ibuprofen (Fig. 5: 11), which were also higher than the particles produced by SAS method. The micrographs from Fig. 5 also indicate the morphology of the particles formed, with several assays producing spherical like particles, and also the presence of filaments in the samples obtained in assay 9 (processed using the higher solution flow rate of 3 mL/min), due to the process of particle formation

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in the feed tube nozzle of the precipitation chamber due to a high ratio between feed solution and CO2 flow rates. Otherwise the lyophilization method produced particles with different shapes (needle-like particles). Table 2 presents results for the estimated diameter of particles obtained at the different precipitation conditions tested, for all assays performed. According to the results, the SAS process reduced the original particle size of sodium ibuprofen from 144 ± 87 μm to 87–264 nm. According to the values from Table 2, no influence of operational pressure was detected in the particle size (assays 1–3). The same behavior was also observed by Martín et al. [32] varying pressures from 100 to 120 bar, using the SAS process with ethanol as primary solvent. Smaller particles were observed for precipitation conditions at lower solute concentration, higher solution flow rate and higher temperature, assays 6, 9 and 4–5, respectively. Sodium ibuprofen solution concentration used in the phase equilibrium experiments was 1 mL/mL, therefore, precipitation conditions 1–5, 8 and 9 from Table 2 are also presented in Fig. 3. Assays 1–5 from Table 2 correspond to the CO2 mass fraction of 95.5% (1 mL/min of solution for 1 kg/h of CO2) while assays 8 and 9 correspond to CO2 mass fractions of 91.4% and 87.7%, respectively. From the phase equilibria data (Fig. 3) a larger quantity of precipitated particles at 35 °C was observed, in comparison to the results obtained at 45 °C, while at 55 °C no precipitated particles were detected. This observation indicates that the sodium ibuprofen solubility increases with enhancing temperature. Therefore, at 110–140 bar (pressures applied in the present study) the system is probably above of its “crossover point”, which arises from the fact that, at high pressures the solubility of sodium ibuprofen in CO2 might be dominated by the effect of the increase of the solid vapor pressure when the temperature is increased. This behavior is well explained by several studies related to scCO2 extraction rate at various conditions of temperature and pressure [25]. Average particle diameter results presented in Table 1 show a decrease in particle size with the temperature increase (assays 2, 4–5) while literature indicates that a solubility increase should result in a decrease of nucleation rates and therefore an increase in particle size [32]. In the present case we believe that the absence of total miscibility (mainly at 35 °C) may result in a quick precipitation that may be leading to particle agglomeration. It might happen because CO2 does not need to leave the atomized droplet for precipitation to occur. The whole process where the CO2 starts diffusing into the droplet causing swelling, solvent evaporation and solute supersaturation, allows a rapid nucleation with formation of several nuclei and smaller particles. It might be occurring

Fig. 6. Calorimetry of ibuprofen processed by Supercritical Anti-solvent (SAS) process, lyophilization and of original sodium ibuprofen (non-processed).

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for higher temperatures such as 55 °C and 45 °C where a very low amount of precipitated particles was observed in the phase equilibrium. The influence of the solution flow rate is explained by Martín et al. [32]: when the ratio CO2/solution flow rate is reduced, either by increasing the solution flow rate or by decreasing the CO2 flow rate, the solute solubility is increased in the fluid due to the cosolvent effect of the organic solvent, which promotes slower nucleation kinetics that produces less particles with bigger sizes and more developed crystalline structure. Finally, the lowest estimated particle size achieved in the present study was obtained at the intermediate pressure tested (110 bar), at the lower temperature (35 °C), at lower solution flow rate (1 mL/min) and lower solute concentration (0.5 mg/mL). The lower particle size found for a solute concentration of 0.5 mg/mL (assay 6) when compared to higher concentrations (assays 2 and 7) might be due to the reduction of agglomeration effects due to the lower concentration of solute in the solvent/anti-solvent solution.

As can be observed in Fig. 3, assays 1–3 and 8–9 were performed in a single phase region above the transition isotherm, which is desirable for SAS assays. However, assays 4 and 5 where performed at a pressure/ temperature/anti-solvent content condition where pressure reduction will not result in phase separation. Therefore, in the assays 4 and 5, in single-phase systems, sodium ibuprofen may have been carried out of the system together with the solvent/anti-solvent solution, reducing particle yield. 3.3. Modification on calorimetric properties of ibuprofen particles Calorimetry profiles obtained by the differential scanning calorimeter, for the SAS samples of the processed ibuprofen are shown in Fig. 6. The samples of SAS particles submitted to calorimetric analysis were selected according to the variability of the SEM results (different shapes and sizes — Fig. 5). Also, samples of the lyophilized ibuprofen and the original

Fig. 7. PXRD patterns (a) and Raman spectra (b) of ibuprofen processed by Supercritical Anti-solvent (SAS) process and lyophilization, and of original sodium ibuprofen (non-processed).

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sodium ibuprofen (non-processed) were also analyzed. According to the calorimetric profiles presented in Fig. 6, the results presented in Fig. 6 indicate that the original and the lyophilized sodium ibuprofen show two groups of peaks, one near to 70–110 °C that probably corresponds to hydration of the sample and other next to 180–210 °C that is characteristic of sodium ibuprofen [6]. On the other hand, the SAS samples showed only one peak, displaced from the two of the original sample, located at near 110 °C. According to Pathak et al. [6], calorimetric results of pure ibuprofen (the acid form) show a single peak between 100 and 120 °C. Since the calorimetric properties and the melting temperature of acid ibuprofen are different from those of the sodium salt, it suggests that the SAS process performed in the present work, besides reducing the particle size, also possibly modified the sodium ibuprofen form.

3.4. Powder X-ray Diffractometry (PXRD) and RAMAN Spectroscopy The PXRD pattern of the SAS processed ibuprofen samples, for the lyophilized sample and also for the original sodium ibuprofen (nonprocessed material) is shown in Fig. 7(a). The non-processed ibuprofen and the lyophilized sample show intense characteristic Bragg reflections of Racemic and homochiral sodium ibuprofen crystalline forms, although the lyophilized sample shows also a huge preferential orientation (more intense ones at 30.4° and 34.5°). On the other hand, only few Bragg reflections were observed in the PXRD patterns of the SAS processed samples, and the most intense patterns do not coincide with those of the sodium ibuprofen phases observed for the non-processed and the lyophilized samples. Contrary to the racemic ibuprofen, which is a racemic compound (equal amounts of left- and right-handed enantiomers of a chiral molecule), the racemic sodium ibuprofen forms a conglomerate (termed the γ-form) as well as two polymorphic compounds, α and β, which are less stable monotropes. From the supercooled liquid, a and b crystallized along with the original γ-form. Forms α and β are “enantiotropically” related with a transition temperature between 75 and 113 °C, but can be considered metastable monotropes of the racemic conglomerate, the stable γ-form. Although PXRD unambiguously rules out the possibility of a racemic compound, it cannot differentiate between a racemic conglomerate and a pseudoracemate [33]. Diffraction angles (2θ) of the peaks and the overall patterns are the same for all samples processed by SAS. Small differences in the peak intensities and peak width may be attributed to preferred orientation and to slight differences in crystallinity. In this sense one can point out the samples from assays 2 and 7 (according to Table 1: obtained at 110 bar, 35 °C, 1 kgCO2/h, 1 mLsolution/min and, respectively, 1 and 1.5 mgsolute/mLsolution) as those that presented more crystalline sodium ibuprofen powder particles compared to original non-processed ibuprofen, lyophilized particles and the SAS particles obtained at higher pressure (assay 3 — 140 bar) and higher solution flow rate (assay 8 — 2 mLsolution/min). The spectra of the SAS processed samples, the lyophilized sample and the original sodium ibuprofen (non-processed) are shown in Fig. 7(b). The RAMAN spectrum of the non-processed sample showed the characteristic RAMAN active modes of ibuprofen sodium salt powder [34]. On the other hand, the RAMAN spectrum of the lyophilized sample showed a very poor data where one can hardly identify features at 800 cm−1, 1200 cm−1 and 1600 cm−1. It is quite controversial since the PXRD pattern of the sample lyophilized suggests a highly crystalline sample. RAMAN spectra of the samples processed by SAS are also very poor with a low frequency background increasing and just broad bands centered at about 800 cm−1, 1200 cm−1 and 1600 cm−1. Small differences among samples in the spectra can be attributed to slight differences in crystallinity, as previously mentioned. In this sense, one can point out the sample from assay 2 (code according to Table 1) as the most crystalline sodium ibuprofen powder particles processed by SAS, which

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indicates 1 mgibuprofen/mL, 1 mLsolution/min, 1 kgCO2/h, 110 bar and 35 °C as the best conditions to obtain crystal sodium ibuprofen. 4. Conclusions Micronization of ibuprofen was successfully performed by SAS using CO2 as supercritical solvent. Phase equilibrium data presented liquid– vapor and solid–liquid–vapor transitions and aided the understanding of particle sizes obtained in SAS assays. The estimated particle sizes of the SAS-precipitated ibuprofen were below 380 ± 84 nm for all the conditions tested in the experiments, which means that the size of the original particles was reduced from micrometric to nanometric order. The best SAS operational conditions, producing the lowest estimated particle size, were: 0.5 mgibuprofen/mL, 1 mLsolution/min, 1 kgCO2/h, 110 bar and 35 °C. Besides sodium ibuprofen is freely soluble water, particle size and morphology are important characteristics that also affect bioavailability [32]: smaller ibuprofen particle sizes, or larger surface areas, increase the dissolution rate of the drug, enhancing its bioavailability. Besides reducing ibuprofen particle size, the SAS process appears to change the original sodium ibuprofen into acid ibuprofen form, according to the results from thermal analysis of the processed and non-processed particles. The PXRD and RAMAN results indicated the SAS process at 1 mgibuprofen/mL, 1 mLsolution/min, 1 kgCO2/h, 110 bar and 35 °C as the best condition to obtain ibuprofen particles with higher crystallinity. Finally, the use of a SFE unit with adaptation for the SAS process was successful and is very useful for research laboratories and for multipurpose industries. Acknowledgments The authors wish to thank CNPq, project number 473153/2012-2, and CAPES (project number 23038.007787/2011-70; AUXPE: 2516/ 2011), Brazilian funding agencies, for the financial support and scholarships which sustain this work. The authors also thank the laboratories LCME and PROFI, from the Federal University of Santa Catarina, for the assistance with the SEM and DSC analyses. References [1] World Health Organization, Model Lists of Essential Medicines, 15th ed. World Health Organization, Geneva, 2007. [2] S. Stolnik, L. Illum, S.S. Davis, Long circulating microparticulate drug carriers, Adv. Drug Deliv. Rev. 16 (1995) 195–199. [3] C. Leuner, J. Dressman, Improving drug solubility for oral delivery using solid dispersions, Eur. J. Pharm. Biopharm. 50 (2000) 47–60. [4] S.M. Moghimi, A.C. Hunter, J.C. Murray, Long-circulating and target-specific nanoparticles: theory to practice, Pharmacol. Rev. 53 (2001) 283–287. [5] O. Kayser, A. Lemke, N. Hernandez-Trejo, The impact of nanobiotechnology on the development of new drug delivery systems, Curr. Pharm. Biotechnol. 6 (2005) 3–7. [6] P. Pathak, M.J. Meziani, T. Desai, Y. Sun, Formation and stabilization of ibuprofen nanoparticles in supercritical fluid processing, J. Supercrit. Fluids 37 (2006) 279–286. [7] A.I. Cooper, Polymer synthesis and processing using supercritical carbon dioxide, J. Mater. Chem. 10 (2000) 207–234. [8] I. Kikic, F. Vecchione, Supercritical impregnation of polymers, Curr. Opin. Solid State Mater. Sci. 7 (2003) 399–405. [9] D.L. Tomasko, H. Li, D. Liu, X. Han, M.J. Wingert, L.J. Lee, K.W. Koelling, A review of CO2 Appl. Process. Pol, Ind. Eng. Chem. Res. 42 (2003) 6431–6456. [10] S.P. Nalawade, F. Picchioni, J.H. Marsman, L.P.B.M. Janssen, The FT-IR studies of the interactions of CO2 and polymers having different chain groups, J. Supercrit. Fluids 36 (2006) 236–244. [11] J.X. Jiao, Q. Xu, L.M. Li, Porous TiO2/SiO2 composite prepared using PEG as template direction reagent with assistance of supercritical CO2, J. Colloid Interface Sci. 316 (2007) 596–603. [12] Y. Kang, J. Wu, G. Yin, Z. Huang, X. Liao, Y. Yao, P. Ouyang, H. Wang, Q. Yang, Characterization and biological evaluation of paclitaxel-loaded poly(L-lactic acid) microparticles prepared by supercritical CO2, Langmuir 24 (2008) 7432–7441. [13] J. Jung, M. Perrut, Particle design using supercritical fluids: literature and patent survey, J. Supercrit. Fluids 20 (2001) 179–219. [14] S.D. Yeo, E. Kiran, Formation of polymer particles with supercritical fluids: a review, J. Supercrit. Fluids 34 (2005) 287–308. [15] P.G. Debenedetti, J.W. Tom, S.D. Yeo, Rapid expansion of supercritical solutions (RESS): fundamentals and applications, Fluid Phase Equilib. 82 (1993) 311–318.

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