HHS Public Access
`Author manuscript
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
`
`Published in final edited form as:
`Eur J Pharm Sci. 2011November20; 44(4): 522-533. doi:l0.1016/j.ejps.2011.09.014 .
`
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`Nanoparticle agglomerates of fluticasone propionate in
`combination with albuterol sulfate as dry powder aerosols
`
`Nashwa El-Gendy1·3, Warangkana Pomputtapitak1, and Cory Berkland 1•2·*
`1Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047.
`
`2Department of Chemical and Petroleum Engineering, The University of Kansas, Lawrence, KS
`66047.
`
`3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-suef
`University, Egypt.
`
`Abstract
`Particle engineering strategies remain at the forefront of aerosol research for localized treatment of
`lung diseases and represent an alternative for systemic drug therapy. With the hastily growing
`popularity and complexity of inhalation therapy, there is a rising demand for tailor-made inhalable
`drug particles capable of affording the most proficient delivery to the lungs and the most
`advantageous therapeutic outcomes. To address this formulation demand, nanoparticle
`agglomeration was used to develop aerosols of the asthma therapeutics, fluticasone or albuterol. In
`addition, a combination aerosol was formed by drying agglomerates of fluticasone nanoparticles in
`the presence of albuterol in solution. Powders of the single drug nanoparticle agglomerates or of
`the combined therapeutics possessed desirable aerodynamic properties for inhalation. Powders
`were efficiently aerosolized (-75% deposition determined by cascade impaction) with high fine
`particle fraction and rapid dissolution. Nanoparticle agglomeration offers a unique approach to
`obtain high performance aerosols from combinations of asthma therapeutics.
`
`Keywords
`
`Fluticasone; albuterol; combination therapy; dry powder; aerosols
`
`1. Introduction
`
`Asthma and chronic obstructive pulmonary disease (COPD) are currently treated using
`either nebulizers, pressurized metered dose inhalers or dry powder inhalers (Dalby and
`Suman, 2003; Murnane et al., 2008b; Yang et al., 2008a). A major determinant of aerosol
`deposition in the respiratory tract is the aerodynamic size of particles and the polydispersity
`
`'To whom correspondence should be addressed. The University of Kansas, 2030 Becker Drive, Lawrence, KS 66047. Phone: (001)
`785- 864-1455. Fax: (001) 785- 864- 1454.berkland@ku.edu.
`Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. AB a service to our
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`
`

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`El-Gendy et al.
`
`Page2
`
`(Louey et al., 2004; Pilcer and Amighi, 2010; Pritchard, 2001). Inhaled drugs should ideally
`possess an aerodynamic diameter less than 5 µm. for delivery into the 'deep' lung for local
`therapy or systemic absorption (Weers et al., 2010). Nanoparticles (<0.5 µm.) are more likely
`to be exhaled, which may lead to dose variability (Shi et al., 2007). If delivered as a
`suspension, such small particles are also prone to particle growth due to Ostwald ripening
`and can suffer from uncontrolled agglomeration (Berkland, 2010). A major obstacle to
`inhaled therapeutics is the inability to efficiently deliver large quantities of a drug to the
`deep lung (Gillian, 2010).
`
`Natural aerosols, in particular, spores from molds and fungi as well as soot and asbestos,
`have a size and structure that allows them to aerosolize efficiently into the lungs. They are
`composed of underlying nanostructures that join together to form microparticles. Following
`this rationale, nanoparticle agglomerates were designed by formulating nanometer-sized
`drug particles, then assembling them to micron-sized clusters with the desired aerodynamic
`diameter (e.g., 1 µm. for treating distal airways or 3-5 µ m for treating upper airways)
`(Bailey et al., 2008; El Gendy et al., 2009; Plumley et al., 2009). By agglomerating
`nanoparticles under controlled process conditions, nanoparticle agglomerate dry powders
`can be tailored to the desired physical and chemical characteristics for aerosol delivery and
`dissolution (Aillon et al., 2010; El-Gendy and Berkland, 2009).
`
`Asthma is a disease that is commonly treated with two types of aerosolized agents;
`bronchodilators (fu agonists) and anti-inflammatory agents (steroidal compounds). Apart
`from acute asthma attacks, which are primarily treated with short acting ~2 agonists, there is
`a strong need for chronic therapy to reduce inflammation and to avoid asthma exacerbations
`(Barnes, 2002). Therapeutic interventions using combinations of a ~ agonist and a
`glucocorticoid have emerged as an effective asthma management strategy to control
`persistent asthma (Rajeswari et al., 2006). The use of~ agonists to prevent bronchial spasm
`and glucocorticoids to decrease inflammation is widely accepted (W estmeier and Steckel,
`2008). Combination formulations have also been suggested to be more effective than a
`single drug due to synergistic effects in the same target cell in the lung epithelia. It appears
`rational, therefore, to combine both substances in one particle instead of formulating a
`combination product containing both drugs in a physical mixture (Adi et al., 2008; Nelson et
`al., 2003; Papi et al., 2007).
`
`Combination products such as Advair and Symbicort are currently marketed. Advair
`combines fluticasone propionate and salmeterol xinafoate into one inhaler (Michael et al.,
`2000). Salmeterol (long acting~ agonists) does not replace the need for rescue inhalers,
`such as albuterol, which are still necessary for immediate relief of asthma symptoms (Kamin
`et al., 2007; Salpeter et al., 2006). Symbicort is another combination product containing
`budesonide and fonnoterol. Fluticasone propionate is a synthetic corticosteroid used to treat
`asthma, allergic rhinitis (hay fever) and eosinophilic esophagitis (Murnane et al., 2008a;
`Rehman et al., 2004; Vatanara et al., 2009). Albuterol sulfate is a short-acting P2
`adrenoreceptor agonist used for the relief ofbronchospasm in conditions such as asthma and
`COPD, and is currently one of the most prescribed bronchodilators for the treatment of
`bronchial asthma (Ahmad et al., 2009; Xu et al., 2010).
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
`
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`El-Gendy et al.
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`Development of dry powder aerosols for delivering fluticasone and/or albuterol nanoparticle
`agglomerates as single anti-asthmatic therapies or in combination to achieve synergistic
`effect is herein described. The study illustrates the formulation of fluticasone nanoparticles
`using potentially acceptable surfactants that control the size and surface charge of the
`prepared nanoparticles. Also, albuterol nanoparticles free of excipients were engineered
`using different techniques. The nanoparticle suspensions were destabilized via ionic charge
`interactions using L-leucine. Combination drug formulations were prepared by adding
`albuterol aqueous solution to the fluticasone nanoparticle suspension followed by addition of
`L-leucine. The aerosol performance of these nanoparticles agglomerate formulations were
`fully characterized and compared to micronized stock drug.
`
`2. Materials and methods
`
`2.1. Materials
`
`Fluticasone propionate (Flu) and albuterol sulfate (Albu) were generously provided by 3M .
`L-a-phosphatidylcholine (lecithin; Lee), cetyl alcohol (CA), L-leucine (Leu) and
`polyvinylpyrrolidone K90 (PVP) were purchased from Sigma Chemical Co., USA. Pluronic
`F-127 (PL, Mw-12,220) was purchased from BASF, USA. Ethanol, acetone, potassium
`dihydrogen phosphate (KH2P04), disodium hydrogen phosphate (Na2HP04) and sodium
`chloride (NaCl) were purchased through Fisher Scientific, USA. Floatable dialysis
`membrane units (MWCO=lO kDa) were obtained from Spectrum Laboratories Inc., USA.
`Amicon Ultra Centrifugal filter units (MWC0=5 kDa) used for dissolution were purchased
`from Millipore, Co (Billerica, MA). Double-distilled water was used throughout the study,
`provided by an EASYpure® RODI (Barnstead International, USA).
`
`2.2. Nanoparticle formulation
`
`2.2.1. Preparation of fluticasone nanoparticle suspension-Nanoparticle
`suspensions of fluticasone propionate were prepared using antisolvent precipitation .
`Solutions of the drug in organic solvent (acetone or ethanol) were prepared at different
`concentrations and directly injected into water at a rate of 2.5 rnUmin. A variety of solvent/
`non-solvent ratios were precipitated under ultrasonication (probe-type sonicator, Fisher
`Scientific, Sonic Dismembrator) operating with an amplitude of 48% in an ice bath or under
`homogenization (probe-type homogenizer, Tissue tearor, Biospec Products, Inc.).
`Hydrophobic surfactants (cetyl alcohol and lecithin) were added to the drug solution while
`hydrophilic surfactants (PL F 127, PV A and PVP K90) were dissolved in the aqueous phase.
`
`2.2.2. Formulation of combination therapy-The combined formulation was prepared
`by adding albuterol sulfate dissolved in water to the precipitated fluticasone propionate
`nanosuspension during homogenization at 25,000 rpm. The two drugs were combined, at a
`ratio of2: 1 w/w, fluticasone propionate: albuterol sulfate (Papi et al., 2007; Westmeier and
`Steckel, 2008).
`
`2.2.3. Fabrication of albuterol nanoparticle suspension-Albuterol sulfate
`nanoparticles were prepared by precipitation or by a top-down (attrition) method.
`Concerning the precipitation technique, solutions of albuterol in water were prepared and
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`El-Gendy et al.
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`Page4
`
`directly injected into ethanol or acetone at a rate of2.5 mUmin. Various solvent/ non(cid:173)
`solvent ratios were used under ultrasonication operating with an amplitude of 48% or under
`homogenization. In the top-down method, albuterol nanoparticles were prepared by
`ultrasonicating or homogenizing a suspension of albuterol in acetone or ethanol. The
`concentration of the drug in the anti-solvent was varied between 0.2 and 1 mg/mL. The
`ultrasonication or homogenization time was also varied.
`
`2.3. Characterization of nanopartlcle suspensions
`
`The average size and polydispersities of the nanoparticle suspensions were determined by
`dynamic light scattering (Brookhaven, ZetaP ALS, SA). The same instrument was used to
`determine the zeta potential of the nanoparticles in 1 mM potassium chloride solution. Three
`runs of 15 cycles were acquired, and the mean zeta potential was recorded. Measurements
`were taken at an angle of 90° to the incident light source. Some samples were frozen at -80
`°C and lyophilized (FreeZone 1) for -36 hat a temperature of-50 °C under vacuum (-0.02
`millibar). Lyophilized powder was stored at room temperature for further characterization.
`
`2.4. Agglomeration of nanoparticle suspensions
`
`Nanoparticles were agglomerated via addition of an agglomerating agent. L-Leucine solution
`in water (2.5 mg/mL) was slowly injected into nanoparticle colloids during homogenization
`at 25,000 rpm for 30 s. The amount of L-leucine added was adjusted to a fluticasone: L(cid:173)
`leucine ratio equal to 1 : 1 for agglomerating the fluticasone suspension and the combination
`suspension. An albuterol: L-leucine ratio of 1: 1.5 was used for agglomerating the albuterol
`suspension.
`
`The agglomerated suspensions were incubated with the agglomerating agent for three hours.
`Then, the size of the prepared nanoparticle agglomerates was measured in Isoton diluent
`using a Coulter Multisizer 3 (Beckman Coulter Inc.) equipped with a 100 )JIIl aperture. The
`suspensions were kept overnight at room temperature to allow evaporation of organic
`solvent and then frozen at -80 °C. The frozen suspensions were transferred to the freeze
`dryer where drying lasted for -3 days. Lyophilized powder was stored at room temperature
`for further characterization.
`
`2.5. Particle size and morphology by transmission electron microscopy {TEM)
`
`Lyophilized powders were resuspended in Isotonic solution and the particle size and size
`distribution was detected using a Coulter Multisizer 3. In addition, the size and morphology
`of the lyophilized nanoparticles and nanoparticle agglomerate powders were evaluated using
`JEOL 1200 EXII transmission electron microscope. Prior to imaging, carbon-coated grids
`(Electron Microscopy Sciences) were placed on a droplet of the suspensions on a glass
`microscope slide to permit the adsorption of the particles onto the grid. After this, the grid
`was blotted with a filter paper and air dried for 1 h .
`
`2.6. Powder flow characteristics
`
`Nanoparticle agglomerate dry powders were poured through a glass funnel from a height of
`4 cm onto a level bench top. The angle that the side of the conical heap made with the
`horizontal plane was recorded as the angle of repose (tan e =height I radius). In addition,
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`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`El-Gendy et al.
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`bulk and tapped densities were determined. Then, the Hausner ratio (tapped density I bulk
`density) and Carr's compressibility index (CJ [(tapped density- bulk density)/ tapped
`density X 100%] were calculated (Kumar et al., 2001; Louey et al., 2004).
`
`2.7. Evaluation of Aerosol performance of nanoparticle agglomerate dry powders
`
`2.7.1. Measurement of theoretical mass mean aerodynamic diameter-The
`geometric particle size and tap density measurements were used for calculating the
`theoretical mass mean aerodynamic diameter (dae) of the nanoparticle agglomerates (El(cid:173)
`Gendy et al., 201 Ob; Fiegel et al., 2008).
`
`2.7.2. Aerodynamic size distribution by time-of-flight analysis-The aerodynamic
`diameter and size distributions of the nanoparticle agglomerate powders were determined by
`time-of-flight measurement (TOP) using an Aerosizer LD (Amherst Instruments, Hadely,
`MA, USA) equipped with a 700 µm aperture operating at 6 psi. For these studies, -1 mg of
`the powder was added to the instrument disperser and data were collected for -60 s under
`high shear force (-3.4 kPa). The instrument size limits were 0.10--200 µm and particle
`counts were above 100,000 for all measurements.
`
`2.7.3. In vitro aerosol deposition of nanoparticle agglomerates by cascade
`impactor-An eight-stage Mark II Andersen Cascade Impactor (ACI, Tisch
`Environmental, Inc.) had stages with particle aerodynamic diameter specifications at a flow
`rate of28.3 Umin as follows: pre-separator (10.00 µm), stage 0 (9.00 µm), stage 1 (5.80
`µm), stage 2 (4.70 µm), stage 3 (3.30 µm), stage 4 (2.10 µm), stage 5 {l.10 µm), stage 6
`(0.70 µm), stage 7 (0.40 µm) and the final filter(< 0.40 µm). Aerodynamic behavior of
`nanoparticle agglomerate dry powders was assessed using the ACI and compared with that
`of the two drugs as received.
`
`The powder was delivered into the cascade impactor by placing capsules (gelatin type, size
`3, generously provided from Capsugel®, NJ, USA) containing 5 ± 0.5 mg of powder into a
`Plastiape Monodose Inhaler RSOl Model 7. The capsule was punctured and the powder was
`drawn through the cascade impactor which was operated at a flow rate of 28.3 Umin for 4 s.
`Dry powder aerosols deposited on each of the nine stages of the impactor were quantified by
`HPLC. After actuation, the device, capsule, adapter, throat, all plates, stages and filter were
`washed into separate volumetric flasks using ethanol (for fluticasone alone or Flu/Alu
`combination) or phosphate buffered saline (pH 7.4 for albuterol). Appropriate sample
`dilutions were made prior to testing by HPLC. Each sample was tested in triplicate.
`
`Concerning the combination formula, the powder deposited on stages was suspended in
`ethanol and was ultrasonicated in a bath-type sonicator (Branson 3510) for 30 min. Then,
`the solution was centrifuged (Beckman, Avanti) at-15,000 rpm for 30 min and the amount
`of fluticasone in the supernatant was determined using a reversed-phase HPLC method. As
`albuterol has a very slightly solubility in ethanol, the drug content in both supernatant and
`precipitate were detected by HPLC.
`
`All ACI experiments were performed under controlled conditions (21 ± 2 °C, 50 ± 5% RH)
`in triplicate. The emitted dose (ED) was defmed as the mass of drug delivered from the
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`El-Gendy et al.
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`Page6
`
`inhaler (i.e., total amount excluding the inhaler device and capsule) (Xu et al., 2010). The
`emitted fraction was determined as the percent of the emitted dose divided by the initial
`mass delivered into the impactor (Lechuga-Ballesteros et al., 2008; Shur et al., 2008; Yang
`et al., 2008b ).
`
`The fine particle fraction of the total dose (FPFm) was calculated as the percentage of
`aerosolized particles that reached the lower seven stages of the impactor (corresponding to
`aerodynamic diameters below 5 µm; stage 2-filter), or the lower five stages (corresponding
`to aerodynamic diameters below 3 µm; stage 4-filter) (El-Gendy et al., 2010a).
`
`The fine particle fraction of the emitted dose (FPFED) was determined from the cumulative
`mass distribution curve at 5 µm and 3 µm and was calculated as a function of the emitted
`dose. Additionally, mass median aerodynamic diameter (MMAD) and geometric standard
`deviation (GSD) were determined from the cumulative mass distribution curve (Pham and
`Wiedmann, 2001; Vanbever et al., 1999; Xu et al., 2010).
`
`2.8. Solid-state characterization
`
`2.8.1. Power X-Ray Diffraction (PXRD)-For analysis of crystallinity, X-ray diffraction
`analysis was performed using an XGEN-4000 (Scintag, Inc.). The powders were analyzed
`over the range of5° to 45° (20) at 45.0 kV and 35.0 mA.
`
`2.8.2. Differential scanning calorimetry (DSC)-DSC data of materials as received,
`nanoparticles and nanoparticle agglomerates were collected using a Q 100 DSC from TA
`Instruments. For thermogram acquisition, sample sizes of 1 to 5 mg were weighed into
`aluminum hermetic pans. Measurement was carried out under inert conditions (nitrogen
`flow of 50 mL/min) with a scan rate of 10 °C/min from 25 to 350 °C.
`
`2.8.3. Thermogravimetric analysis (TGA)-TGA was also performed using a Q50
`TGA from TA Instruments. A platinum sample pan was loaded with 5 ± 0.5 mg of sample
`and heated from 25 to 350 °C at a rate of 10 °C/min under dry nitrogen flowing at rate of 40
`mL/min. Data analysis was completed using Universal Analysis 2000 (Version 4.3A)
`software that was provided by TA Instruments.
`
`2.9. Determination of process yield
`
`The weight of dry powder for the prepared nanoparticle agglomerates was measured and the
`yield was calculated (El Gendy et al., 2009).
`
`2.10. Determination of drug content uniformity of nanoparticle agglomerates
`
`Fluticasone content in the dry powders was assessed by dispersing 1 mg of the lyophilized
`powder in 10 mL of ethanol. The dispersion was ultrasonicated in a bath-type sonicator for
`30 min. Then the solution was centrifuged at -15,000 rpm for 30 min to remove insoluble
`ingredients and the amount of fluticasone in the supernatant was determined using a
`reversed-phase HPLC method. For the combination formula, albuterol content was detected
`in both the pellet after dissolving in 10 mL PBS and in the supernatant using HPLC. Drug
`content in albuterol nanoparticle agglomerates was determined by dispersing 1 mg of the
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`Page?
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`lyophilized powder in PBS. The dispersion was ultrasonicated in a bath-type sonicator for
`15 min. Then the amount of albuterol was determined using a reversed-phase HPLC method.
`All experiments were performed in triplicate and drug content was calculated (El Gendy et
`al., 2009).
`
`2.11. Dissolution studies
`
`The dissolution of the prepared nanoparticles and nanoparticle agglomerates was determined
`under sink condition and compared with the dissolution of the drugs as received. The
`dissolution offluticasone was carried out at 37 ± 0.5 °C in a 1 liter beaker. Lyophilized
`powder (-10 mg) was dispersed in 10 mL PBS (pH 7 .4) and was suspended in a floatable
`dialysis membrane unit (Mw cut-off= 10 kDa). The unit was allowed to float in 500 mL of
`PBS and the whole assembly was stirred at a constant speed (100 rpm) using a magnetic
`stirrer (Barnstead, Thermolyne MIRAK™). At predetermined time intervals for a total
`period of8 h, samples (1 mL) of the medium were withdrawn from the dialysis bag and
`replaced with fresh medium. Then, the samples were centrifuged for 30 min at -13,000 rpm.
`The supernatant was removed and the pellet was dissolved in 1 mL of ethanol. Fluticasone
`content was determined using reversed-phase HPLC. In the case of the combination
`formula, both pellet and supernatant were analyzed for albuterol using a reversed-phase
`HPLC method. Studies were conducted in triplicate.
`
`For albuterol dry powders, 5 mg was dispersed in 0.6 mL phosphate buffered saline (PBS,
`pH 7.4) and placed in a 5 kDa Ultra Centrifugal filter unit which was immersed inside a 10
`mL centrifugal tube containing PBS solution to a final volume of7 mL. All samples were
`incubated at 37 ± 0.5 °C and shaken at 100 rpm. One mL aliquots were taken at various time
`points up to 8 hours from the bulk solution and replaced with 1 mL of fresh PBS. The drug
`concentration was measured using reversed-phase HPLC. All experiments were performed
`in triplicate.
`
`2.12. Quantitative analysis of the drug concentrations by HPLC
`
`Drug content, dissolution, and ACI concentration on stages were carried out using reversed(cid:173)
`phase HPLC methods. A Shimadzu HPLC system including a solvent delivery pump
`(Shimadzu LC-lOAT), a controller (Shimadzu SCL-lOA), SIL-lOAxL autoinjector, and a
`SPD-lOA UV detector was used in this study. Chromatograms were acquired and analyzed
`using Shimadzu Class VP 4.3 software. A long Zorbax SB C-18 column (Agilent C; 4.6 mm
`x 100 mm) with a particle diameter of3.5 µm was used for separation. During the assay of
`fluticasone, the drug was eluted isocratically at a mobile phase flow rate of 1.2 mL/min and
`monitored with a UV detector operating at 238 nm. The mobile phase for the assay consisted
`of an acetonitrile and water mixture (65 :35 v/v) (Asmus et al., 2004; Steckel and Muller,
`1998). The run time for the assay was 10 minutes, and the retention time for fluticasone was
`3.9 ± 0.2 min. For analyzing albuterol samples, an isocratic system was used with mobile
`phase of 90: 10 v/v phosphate buffer (10 mM, pH3 .5): acetonitrile at a flow rate of 0.3
`mUmin and detection was performed at 225 nm (Kamin et al., 2007). The run time for the
`assay was 10 min, and the retention time for albuterol was 3 .85 ± 0.4 min.
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
`
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`3. Results and discussion
`
`3.1. Fluticasone nanopaticle suspensions made by precipitation
`
`To obtain small nanoparticles from poorly water soluble fluticasone propionate, a non(cid:173)
`solvent precipitation method was employed (Bilati et al., 2005). Selected surfactants were
`chosen from a list of excipients that may be appropriate for inhalation (Chougule et al.,
`2007; Pilcer and Amighi, 2010). Different suspensions were produced using water as anti(cid:173)
`solvent and a drug concentration ofO.l % or 0.2% w/w dissolved in ethanol or acetone.
`Fluticasone particles prepared without surfactants were very large with high polydispersity.
`Mean diameters of fluticasone nanoparticles made with individual surfactants were larger
`than those with combined surfactants. Particle size tended to increase and colloidal stability
`was difficult to maintain as fluticasone concentration was increased. Nanoparticles
`precipitated from acetone were larger than those precipitated from ethanol. Nanoparticle size
`appeared to decease slightly when using ultrasonication rather than homogenization during
`the precipitation process (Supplementary Table 1).
`
`The most successful fluticasone nanosuspension was prepared by precipitation from ethanol
`underultrasonication (0.1% w/vFlu + 0.01% w/v PVP + 0.005% w/v Lee). The surfactant
`combination employed yielded a small drug particle size (-400 nm) and low polydispersity
`(0.132). The charged surface of the nanoparticles (-12 m V) allowed the potential to
`destabilize this colloid via interaction with an agglomerating agent (Table 1 ). This formula
`was chosen for the preparation of the fluticasone nanoparticle agglomerates and for the
`combination formulation with albuterol sulfate in solution.
`
`3.2. Albuterol nanoparticle suspensions made by two approaches
`
`3.2.1. Production of nanoparticles by precipitation-Albuterol nanoparticles were
`first prepared by precipitation. A smaller nanoparticle size was produced when using
`acetone as non-solvent as opposed to ethanol. A decrease in particle diameter followed from
`a decrease of the drug concentration. The smallest nanoparticle size was obtained at a 2.5/25
`water/acetone ratio at a drug concentration of0.1 % w/v; however, the polydispersity was
`high with low yields for all precipitation trials (Supplementary Table 2).
`
`3.2.2. Production of nanoparticles by attrition-Nanoparticles were also produced
`by fragmenting micronized drug particles using homogenization or ultrasonication. The
`homogenizer was superior to ultrasonication in the preparation of albuterol nanoparticles
`causing a significant decrease in size with low polydispersity (Supplementary Table 3). With
`these considerations in mind, the A 7 nanoparticle formulation was selected which was
`prepared by homogenizing a suspension of the drug in acetone in a concentration of 1
`mg/mL for 15 min (Table 1).
`
`3.3. Agglomeration of the formulated nanoparticles
`
`Colloidal suspensions offluticasone nanoparticles, albuterol nanoparticles (A7) and
`fluticasone nanoparticles combined with albuterol in solution were destabilized using L(cid:173)
`leucine to disrupt the electrostatic repulsion between particles (Young et al., 2002). The
`resulting nanoparticle agglomerates had a geometric size of-3-5 }Utl (Table 2). After
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`El-Gendy et al.
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`Page9
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`drying, powders showed a slightly broader size distribution (Fig. 1 ). In addition, the
`combination powders had a wider distribution compared to the single-drug formulations,
`perhaps due to large albuterol particles formed during freeze drying.
`
`The particle size of the nanoparticles and nanoparticle agglomerates was congruent with the
`structures observed in TEM micrographs. Fluticasone nanoparticles were slightly elongated
`with smooth surfaces and a particle size of -400 run (Fig. 2A). Nanoparticle agglomerates
`appeared as elongated nanoparticles that were agglomerated together into micron-sized
`clusters with a somewhat porous structure (Fig. 2B). The combination powders of
`fluticasone nanoparticles dried with albuterol in solution generally exhibited slightly larger
`particles with a rough surface. Presumably, albuterol in solution deposited on the rod-shaped
`fluticasone particles during drying (Fig. 2C). Small albuterol nanoparticles with a particle
`diameter less than 100 run were (Fig. 3A) [/agglomerated into micron-sized particles (Fig.
`3B).
`
`Powder properties for micronized drugs as received and nanoparticle agglomerates were also
`studied. Flowability and density characterization helped elucidate any differences in bulk
`powder properties (Table 3). Flowability indices were calculated from density differences
`and the angle of repose. The micronized drugs showed a larger angle of repose, greater tap
`density and higher values of the Hausner ratio and Carr's index compared to the
`nanoparticle agglomerates (Fig. 4). This was probably the result of a reduction of cohesive
`forces in nanoparticle agglomerates compared to drug powders as received. L-Leucine may
`have also reduced surface energy in nanoparticle agglomerate dry powders (Shur et al.,
`2008).
`
`3.4. Nanoparticle agglomerates yielded desirable aerosol characteristics
`Theoretical mass mean aerodynamic diameters ( daem) of the prepared nanoparticle
`agglomerates were calculated from the geometric particle size and tap density. The
`calculated daero (0.8--1. l µm) was appropriate for increasing the probability of aerosol
`deposition in the alveolar region of the lungs. Aerosizer LD time-of-fight (TOF)
`measurements of nanoparticle agglomerate dry powders also showed particles in the
`respirable size range (2 - 3.2 µm) with relatively narrow size distribution (Table 2 and Fig.
`5). The MAD value of the combination formula obtained by Aerosizer appeared to be close
`to that of the pure fluticasone nanoparticle agglomerates.
`
`Cascade impactor analysis is the standard technique for in vitro characterization of dry
`powder aerosols. Fluticasone nanoparticle agglomerates mainly deposited on stages 3 and 4,
`while albuterol nanoparticle agglomerates favored deposition on stages 4 and 5 of the
`impactor. The combination formula exhibited size distribution similar to fluticasone
`nanoparticle agglomerates with some additional powder deposition on upper stages. This
`may be explained by the fact that once fluticasone nanoparticle agglomerates were dried in
`the presence of albuterol in solution, forces such as van Der Waals are sufficient to hold all
`particles together. This may suggest that fluticasone nanoparticle agglomerates were indeed
`a 'carrier' for much of the albuterol. Albuterol in the combination showed a different
`deposition profile than the fluticasone, although the albuterol deposition was improved
`
`Eur J Phann Sci. Author manuscript; available in PMC 2015 September 25.
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`El-Gendy et al.
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`Page 10
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`compared to the albuterol as received. This may be due to the segregation of some larger
`albuterol particles from the fluticasone nanoparticle agglomerates.
`
`Conversely, drug powders as received largely deposited on the mouthpiece, throat and the
`upper stages (Fig. 6). A larger percentage of fluticasone (27%) and albuterol (28%) powder
`as received remained in the capsule shell and device when compared to the nanoparticle
`agglomerates (9% for Flu NA, 11 % for Albu NA and 18% for the combined formulation).
`This reflected the efficient aerosolization and high fine particle fraction of the nanoparticle
`agglomerates. In addition, the irregular shape of the nanoparticle agglomerates may decrease
`their contact area with device su