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MATERIALS AND INTERFACES Preparation of NaCl Powder Suitable for Inhalation Patricia Tang,†,‡ Hak-Kim Chan,*,‡ Esther Tam,§ Nike de Gruyter,| and John Chan⊥ Department of Chemical Engineering and Faculty of Pharmacy, UniVersity of Sydney, NSW 2006, Australia, Department of Chemical Engineering, UniVersity of Western Ontario, Ontario, Canada, Faculty of Pharmacy, UniVersity of Utrecht, 3508 TC Utrecht, The Netherlands, and North Sydney Boys High School, North Sydney, NSW 2060, Australia
The use of aerosolized hypertonic saline (4.5% NaCl) as an osmotic agent for bronchial provocation tests to identify people with asthma is problematic due to the inconvenience in administration using ultrasonic nebulizers. Delivery using dry powder inhalers is an attractive solution but production of inhalable powders of NaCl is challenging because of physical stability issues. In this study, dry powders of NaCl were prepared using an ultrasound-mediated precipitation process followed by spray drying. The resulting particles (mass median diameter, 2.12 ( 0.35 µm) showed the typical cubic crystal habit of NaCl and were confirmed to be crystalline by X-ray powder diffraction. Dispersion of the particles by dry powder inhalers (Inhalator, Dinkihaler, and Aeroliser) showed aerosol performance (fine particle fraction, 18, 35, and 36%, respectively) suitable for the bronchoprovocation test. The present method can thus be a promising alternative for preparing dry powder NaCl aerosols for inhalation delivery. 1. Introduction Nebulized aerosols of 4.5% (w/v) sodium chloride as an osmotic agent have been used widely for bronchial provocation tests to identify people with active asthma or exercised-induced asthma.1-4 When sodium chloride is inhaled into the airways, the osmolarity of the fluid lining the mucosal surfaces is increased. The bronchial muscle of an asthmatic person will contract with response to the rate of change of osmolarity. The change in osmolarity also induces mucous clearance and therefore is beneficial for patients suffering from cystic fibrosis and chronic bronchitis. The use of nebulizers, however, becomes increasingly unpopular because of their large size and the longer time taken to deliver the aerosols compared to dry powder inhalers (DPIs). Due to the limitations of nebulizers, it would be beneficial if sodium chloride powder suitable for inhalation can be produced. Anderson et al. have proven that a dry powder of sodium chloride could potentially replace the nebulized form to assess bronchial responsiveness in patients with asthma.4 However, particles produced by conventional crystallization process are too large to inhale. Previous methods to produce an inhalable form of NaCl powder is by micronization (milling twice) followed by vacuum-drying in an oven5 at 176 °C for approximately 1 h. Thermal annealing is necessary to lower the energy state and thereby reduce aggregation of the physically unstable milled particles. This method is tedious, and other suitable production methods are thus desirable (e.g. spray drying, which has been used successfully for other drugs). However, * To whom correspondence should be addressed. Tel.: +61 2 9351 3054. Fax: +61 2 9351 4391. E-mail:
[email protected]. † Department of Chemical Engineering, University of Sydney. ‡ Faculty of Pharmacy, University of Sydney. § University of Western Ontario. | University of Utrecht. ⊥ North Sydney Boys High School.
particles obtained from spray drying of NaCl solution are highly unstable and undergo extensive recrystallization, forming large agglomerates which are difficult to disperse and hence unsuitable for inhalation. The present work reports on an alternative method to prepare stable NaCl powder suitable for inhalation as an osmotic agent. Our method involves precipitation of NaCl particles under the action of ultrasonic waves. The ultrasonic waves help to nucleate and crystallize NaCl particles more evenly and faster. Qian et al. reported that the conversion of precursor to ZnO nanocrystals was completed in just 12 min with sonification compared to 12 days without it.6 Nucleation and crystal growth process can be achieved in a short time because ultrasound reduces the width of the metastable zone and hence nucleation can start at a lower level of supersaturation.7 Sonication process is based on cavitation resulting from constant creation, growth, and implosive disintegration of bubbles in liquid.8 Apart from the fast procedure, sonication also offers further advantages: (i) it produces smaller crystal size and narrower size distribution of the products than a conventional crystallization process because crystal growth occurs at a lower supersaturation level where initial growth is less rapid, (ii) it is relatively cheap, (iii) the process can be done at ambient conditions, and (iv) the reaction vessel required is relatively small and simple shaped, which makes the cleaning process easy for the pharmaceutical requirements.7 Various parameters affecting the process including NaCl concentration, the volume of the antisolvent, sonification time, sonic waves output power and frequency, and stirring rate were investigated. The effect of surfactant, sodium lauryl sulfate (SLS), on the particle size was also studied. The recovery of the precipitated NaCl powder as free flowing powder was attempted via a number of separation/drying techniques, namely, centrifugation, filtration, evaporation, and spray drying.
10.1021/ie051414+ CCC: $33.50 © 2006 American Chemical Society Published on Web 05/12/2006
Ind. Eng. Chem. Res., Vol. 45, No. 12, 2006 4189 Table 1. Effect of Experimental Parameters on NaCl Particle Size (Overhead Stirrer at 100 rpm)a exp ID
ethanol vol (mL)
NaCl (g/mL)
NaCl added (mL)
EtOH/NaCl (mL/mL)
ultrasonic time (min)
D(V,0.5) (µm)
span
1 2 3 4 5 6 7 8
200 200 200 300 300 300 350 400
0.10 0.10 0.10 0.10 0.20 0.15 0.15 0.15
4 4 4 4 4 4 4 4
1081.5 1081.5 1081.5 1622.3 811.1 1081.5 1261.8 1422.0
5 10 20 5 5 5 5 5
2.01 ( 0.11 2.32 ( 0.09 2.14 ( 0.10 1.98 ( 0.12 2.04 ( 0.24 2.06 ( 0.26 2.08 ( 0.31 2.56 ( 0.29
1.72 ( 0.32 3.71 ( 0.21 2.57 ( 0.27 1.69 ( 0.26 1.77 ( 0.25 1.68 ( 0.25 1.76 ( 0.23 1.98 ( 0.25
a
A constant frequency of 40 kHz and power of 50 W were used.
2. Method 2.1. Ultrasonic Precipitation. A concentrated NaCl solution was introduced into ethanol as the antisolvent at a known volume ratio between 1:50 and 1:100 in a 500 mL beaker (diameter, 9 cm). An ultrasonic bath or probe was used to provide sonic waves to enhance micromixing. The ultrasonic bath (Unisonics, NSW, Australia) has a frequency of 40 kHz and output power of 50 W, while the probe (Model 250, Branson Ultrasonics Corp., Danbury, CT) has a lower frequency of 20 kHz with an adjustable power output. An overhead stirrer (50300 rpm) was also employed to provide macroscopic mixing. To investigate the effect of surfactant, 0.6 mL of 33%w/w of sodium lauryl sulfate (SLS) solution was added to the ethanol before NaCl solution was introduced. Each experiment was repeated in triplicate to ensure reproducibility. 2.2. Drying. A rotary evaporator (Rotavapor, Buchi, Switzerland), a centrifuge (Jouan CT422, John Morris Proprietary Ltd., NSW, Australia), and a spray dryer (Buchi B-191, Flawil, Switzerland) were tested for their suitability to facilitate the production of dry NaCl powder from the suspension. The first technique involved filtering NaCl suspension through a Nylon membrane (pore size, 0.2 µm), resuspending the cake in 2-propanol, and evaporating the solution at approximately 85 °C using a conventional rotary evaporator. In the second technique, centrifugation of the NaCl suspension was employed at room temperature using speeds of 1500, 3500, and 4000 rpm. In the last drying technique, the NaCl suspension was spraydried under the following conditions: inlet temperature, 90 °C; atomizer, 550 L/h; aspiration, 57.6 m3/h; feed inlet, 4.40 ( 0.47 mL/min; outlet temperature, 55-62 °C. During spray drying, the suspension was continually stirred at 1000 rpm. 2.3. Particle Size Measurement. The sizes of NaCl particles after precipitation and spray drying (followed by redispersion in 2-propanol) were determined by laser diffraction (Malvern Mastersizer S, Worcs, U.K.). The size reported is D(V,0.5), which is the equivalent volume diameter at 50% cumulative volume. The width of the size distribution is expressed as span, calculated as [D(V,0.9) - D(V,0.1)]/D(V,0.5). Particle size analysis was based on the refractive index (RI) of NaCl (1.5442), RIimaginary of NaCl (0.03), and RI of 2-propanol as the dispersion liquid (1.378). To ensure full dispersity of the particles, surfactant poly(oxyethylene) sorbitan monooleate (0.2% (v/v)) was added and the NaCl suspension was sonicated for 5 min. Measurement was done in triplicate to ensure reproducibility. Student t-test was performed with a probability of less than 0.05 considered statistically significant (Microsoft Excel version 2003). 2.4. Powder Dispersion. Aerosol performance of the NaCl powders was tested using a multistage liquid impinger (Copley, Nottinghamshire, U.K.) setup as specified in the British Pharmacopeia.9 The DPIs used in this work to test the dispersion of sodium chloride were Dinkihaler (Aventis Pharma, Bridgewater,
NJ), Inhalator (Boehringer Ingelheim, Ingelheim, Germany), and Aeroliser (Novartis Pharmaceuticals, Sydney, Australia). Dinkihaler has a low resistance to airflow, allowing high inspiration flow rates greater than 60 L/min, while Inhalator has a high resistance which normally allows inspiratory flow rates between 30 and 60 L/min. Aeroliser, having a resistance similar to Dinkihaler, was also tested because it is one of the DPIs available in the Australian market that can deliver a large powder dose. Approximately 20 mg of the powder was loaded to a gelatine or hydroxypropyl methylcellulose (HPMC) capsule and dispersed using the DPI into the multistage liquid impinger (MSLI). A minimum of 3 capsules was used for each dispersion test, and the test was performed in triplicate to obtain mean values. Depending on the inhaler, the MSLI was run at two different flow rates (60 and 120 L/min) to maximize powder dispersion using a high flow achievable by the patients. HPMC capsules were used for runs at 120 L/min due to shattering of the gelatine capsules at high flow rate, as found by Chew and Chan.10 NaCl was assayed by vapor pressure osmometry (Knaur model no. 11.00, Berlin, Germany) at 37 °C with deionized water as the reference. A calibration curve was constructed using standard solutions of NaCl: concentration (mg/mL) ) 0.019 × instrument signal (mV) + 0.04 (R2 ) 0.994). In this study, the fine particle dose (FPD) was defined as the mass of particles smaller than 7 µm. The fine particle fraction (FPF) was calculated as the fraction of FPD referenced against the total dose loaded into the device. FPD and FPF were obtained by interpolation to the cumulative mass and percent undersize at aerodynamic diameter of 7 µm. 3. Results 3.1. NaCl Precipitation. Table 1 shows that changing the ultrasonic time from 5 to 10 min (experimental identification nos. (exp ID) 1-2) increased the particle size slightly (p < 0.05, t-test). Qian et al. observed an increase of the ZnO crystal size from 4.7 to 6.4 nm when sonicated for 120 min.6 It was postulated that by increasing the sonication time, the crystals grew due to local heating arising from cavitation. When the sonication time was further increased from 10 to 20 min (exp ID 2-3), the particle size was not statistically different (p > 0.05). It seems that after 10 min, the solute has been depleted and the supersaturation level is too low for the crystals to grow. For the range of ratio of ethanol to NaCl investigated, changing the volume of ethanol and NaCl (exp ID 1, 4-8) does not change the particle size significantly (p > 0.05). To investigate the effect of stirring, 4 mL of 0.1 g/mL NaCl solution was added to 300 mL of ethanol and sonicated for 5 min. This condition was chosen because Table 1 shows slightly smaller particle size with narrower distribution at this condition (exp ID 4) compared to others. Ultrasound waves provide
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Figure 3. Effect of the power (amplitude) of ultrasonic wave on the bubble size and the cavitation energy intensity at a fixed frequency. Panel a represents a lower power than b. Table 3. Effect of Centrifugation Speed and Time on Particle Size (Original Size before Centrifugation, 1.95 ( 0.12 µm)
Figure 1. Effect of stirring rate on particle size (sonication time, 5 min; 300 mL of ethanol; 4 mL of NaCl (0.1 g/mL). A constant frequency of 40 kHz and power of 50 W were used.
centrifugation speed (rpm)
centrifugation time (min)
D(V,0.5) (µm)
increase in particle size with respect to original particle size (%)
4000
1 3 5 10 15 20 10 15 20
2.78 ( 0.24 3.06 ( 0.19 3.25 ( 0.22 3.32 ( 0.45 3.09 ( 0.19 3.64 ( 0.21 2.92 ( 0.32 2.79 ( 0.28 3.10 ( 0.21
42.6 56.9 66.7 70.3 58.5 86.7 49.7 43.1 59.0
3500 1500
Table 4. NaCl Particle Size before and after Spray Drying after spray drying
D(V,0.5) (µm) span
Figure 2. Effect of the frequency of ultrasonic wave on the number of bubbles per unit volume per unit time (cavitation density) at a given power. Panel a represents a lower frequency than b.
microscopic mixing from vibration, cavitation, and travel of the wave, where the precipitant molecules can be distributed uniformly and rapidly across the solution molecules. As a result, it avoids excessive local supersaturation. To provide macroscopic mixing, mechanical stirring was used here. A slower stirring rate produced smaller particles (Figure 1). No stirring at all actually produced slightly larger particles. This implies that a combination of macromixing and micromixing can help to distribute precipitant molecules more uniformly and lower the supersaturation level. If the stirring rate is too fast, it causes a vortex and channeling in the solution which hinders the formation of bubbles in the liquid medium (i.e. cavitation). As a result, the mixing efficiency is reduced since the formation and collapse of the bubbles, which are responsible for the effective mixing, are affected. Because 50 rpm was found to be the optimal stirring rate (Figure 1), it was used to examine the effect on particle size due to the frequency and intensity of the ultrasonic wave (Table 2). Other experimental conditions were fixed at 300 mL ethanol, 4 mL NaCl at 0.15 g/mL, and 5 min sonication. Frequency of ultrasonic wave affects the amount of cavitation (i.e. creation of bubbles) per unit volume per unit time and
before spray drying
without SLS
0.6 mL of 33% (w/w) SLS
1.86 ( 0.15 1.95 ( 0.22
5.16 ( 1.21 2.89 ( 0.82
2.12 ( 0.35 2.02 ( 0.09
bubble size. The bubble radius is inversely proportional to the frequency as follows:11
Ro )
1 2πf
x
Po F
3γ
(1)
where Ro is the bubble radius, f the vibration frequency, Po the hydrostatic pressure in atmospheres, F the density of liquid, and γ the specific heat ratio of the gas. At a given power, the amount of cavitation increases with increasing frequency, but the bubbles are smaller (Figure 2). Their implosions result in lower energy released because bubble energy is proportional to the square of the bubble radius.12,13 Input power (amplitude) determines the size of the bubbles created.11 At a constant frequency, higher power (amplitude) will create bigger bubbles (Figure 3). When these bubbles burst, the intensity of the cavitation energy released is higher. Table 2 shows that at approximately the same power (55 and 50W), higher frequency (40 kHz) generated smaller particles with narrower size distribution. This is because at higher frequency, although the cavitation energy intensity is low, the number of cavitation implosions per unit volume per unit time is high.14 This could possibly lead to the enhancement of localized micromixing and hence narrow distribution of smaller particle size. At a fixed frequency of 20 kHz, increasing the power from 14 to 33 W did not change the particle size
Table 2. Effect of the Frequency and Power of Ultrasonic Wave on Particle Size (300 mL of Ethanol; 4 mL of NaCl (0.15 g/mL); 5 min Sonication; Overhead Stirrer, 50 rpm) power ) 14 W D(V,0.5) (µm) span
1.71 ( 0.50 1.20 ( 0.11
probe (frequency ) 20 kHz) power ) 33 W power ) 55 W 1.74 ( 0.82 2.80 ( 0.87
3.30 ( 1.73 3.02 ( 1.63
power ) 70 W
ultrasonic bath (f ) 40 kHz) power ) 50 W
3.39 ( 1.86 2.58 ( 1.90
1.40 ( 0.10 1.44 ( 0.17
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Figure 4. (a) Scanning electron micrograph of the NaCl particles and (b) X-ray diffraction pattern of the NaCl particles obtained from precipitation and spray drying.
significantly (Table 2). However, increasing it further to 55 and 70 W enlarged the average particle size and broadened the distribution. When power is too high, the bubbles are bigger and their implosions happened at a larger length scale; hence, the localized micromixing is potentially less effective. 3.2. Drying. When the powder was obtained from filtration and rotary evaporation, there was an increase of 47% of the particle size. The centrifugation was also found unsuitable since there is a large increase in particle size (Table 3). The G-force present during centrifugation seemed to cause the particles to be bonded strongly together. The addition of surfactant, sodium lauryl sulfate (SLS), was ineffective in preserving the original size. Spray drying the NaCl suspension without the presence of SLS as surfactant has led to an increase in the particle size
(Table 4). Adding SLS was found to be effective in reducing the degree of aggregation. The NaCl particles obtained are well-formed with a cubic habit (Figure 4a) and highly crystalline, as shown by the X-ray diffraction pattern (Figure 4b). The highly crystalline NaCl powder obtained from the ultrasonic precipitation is advantageous because it provides stability. Amorphous materials are physically unstable and in high humidity will recrystallize uncontrollably, forming solid bridges between particles which make them unsuitable for inhalation (Figure 5). It is well-known that when milling is employed to produce fine particles, profound changes in crystal structure, with increased content of the amorphous phase, can take place.15 Therefore, this ultrasonic precipitation process has
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sonic waves. The main advantage of this method is that it is rapid and simple and produces small crystal NaCl particles that are crystalline and dispersible. By varying a number of parameters, which include concentration, sonification time, sonic wave output power, and stirring rate, the desired particle size can be achieved. Literature Cited
Figure 5. Scanning electron micrograph of the NaCl particles obtained by spray drying from solution alone. Table 5. Fine Particle Dose (FPD) and Fraction (FPF) Based on Loaded Dose of NaCl Particles Produced by Precipitation and Spray Drying (Particle Size after Spray Drying, 2.12 ( 0.35 µm) inhaler
flow rate (L/min)
FPD (mg)
FPF (%)
Inhalator Aeroliser Dinkihaler
60 120 120
18.0 ( 2.0 36.4 ( 3.6 35.7 ( 3.2
17.9 ( 2.0 35.6 ( 3.5 35.3 ( 3.2
a distinct advantage over the conventional milling in producing crystalline fine particles. 3.3. Dispersion of the NaCl Powder. As the cutoff diameter at each impinger stage varies with flow rate, interpolation of the cumulative undersize plots was used to determine the fine particle dose and fraction. Table 5 shows that the NaCl powders produced from the present method are suitable for inhalation when dispersed using the DPIs. Anderson et al. reported a similar fine particle fraction when NaCl powder, produced by milling, was dispersed using the Inhalator and Halermatic (Fison’s Pharmaceutical Pty Ltd). The percentage of NaCl particles smaller than 7 µm was 15.8 and 30% when dispersed at 60 L/min with Inhalator and Halermatic, respectively. The Dinkihaler and Aeroliser used in the present study have a low resistance and dispersion mechanism similar to the Halermatic.4 Using a maximal inspiratory effort of 80 cm H2O, a patient can generate 150 L/min through the Aeroliser.16 Even with a comfortable inspiratory effort of 40 cm H2O, 105 L/min can still be generated through the Aeroliser. Hence, the results obtained at 120 L/min are realistic for the patient to achieve during the bronchial provocation test. 4. Conclusion Synthesis of NaCl powder suitable for inhalation has been shown to be successful using precipitation under the effect of
(1) Schoeffel, R. E.; Anderson, S. D.; Altounyan, R. E. Bronchial hyperreactivity in response to inhalation of ultrasonically nebulised solutions of distilled water and saline. Br. Med. J. 1981, 283, 1285-1287. (2) Smith, C. M.; Anderson, S. D. Inhalation provocation test using nonisotonic aerosols. J. Allergy Clin. Immunol. 1989, 84, 781-790. (3) Anderson, S. D.; Brannan, J. D.; Chan, H.-K. Use of aerosols for bronchial provocation testing in the laboratory: Where we have been and where we are going. J. Aerosol Med. 2002, 15 (3), 313-324. (4) Anderson, S. D.; Spring, J.; Moore, B.; Rodwell, L. T.; Spalding, N.; Gonda, I.; Chan, K.; Walsh, A.; Clark, A. R. The effect of inhaling a dry powder of sodium chloride on the airways of asthmatic subjects. Eur. Respir. J. 1997, 10, 2465-2473. (5) Clark, A. R.; Hsu, C. C.; Walsh, A. J. Preparation of sodum chloride aerosol formulations; Genentech, Inc.: South San Francisco, CA, 1996; 15 pp, US5747002. (6) Qian, D.; Jiang J. Z.; Hansen, P. L. Preparation of ZnO nanocrystals via ultrasonic irradiation. Chem. Commun. 2003, 9, 1078-1079. (7) Li, H.; Wang, J.; Bao, Y.; Guo, Z.; Zhang, M. Rapid sonocrystallization in the salting-out process. J. Cryst. Growth 2003, 247, 192-198. (8) Suslick, K. S. Sonochemistry. Science 1990, 247 (4949, Pt. 1), 14391445. (9) British Pharmacopeia, Appendix XII, Aerodynamic assessment of fine particles-fine particle dose and particle size distribution, Apparatus C. 2001. (10) Chew, N. Y. K.; Chan, H. K. Influence of particle size, air flow, and inhaler device on the dispersion of mannitol powders as aerosols. Pharm. Res. 1999, 16 (7), 1098-1103. (11) Frederick, J. R. Ultrasonic engineering; John Wiley & Sons: New York, 1965. (12) Piazza, T.; Puskas, W. L. Ideal ultrasonic parameters for delicate parts cleaning. 8th International Symposium on Particles in Surfaces: Detection, Adhesion and Removal, Providence, RI June 24-26, 2002. (13) Puskas, W. L.; Piazza, T. Designer waveforms: Ultrasonic technologies to improve cleaning and eliminate damage. CleanTech 2000, Las Vegas, NV, June 6-8, 2000. (14) Crum, L. A. Acoustic cavitaion series: Part five, Rectified diffusion. Ultrasonics 1984, 22, 215-223. (15) Hosek, P.; Authelin, J. R.; Brown, A. B.; Vemuri, N. M. (RhonePoulenc Rorer Ltd., Great Britain) Milling process for the production of finely milled medicinal substances. PCT Int. Appl. WO2000032165, 2000. (16) Chew, N. Y. K.; Chan, H.-K. In vitro aerosol performance and dose uniformity between the Foradile Aeroliser and the Oxis Turbuhaler. J. Aerosol Med. 2001, 14, p 495-501.
ReceiVed for reView December 19, 2005 ReVised manuscript receiVed March 30, 2006 Accepted April 11, 2006 IE051414+
