Powder Technology 214 (2011) 169176

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Powder Technology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p ow t e c

Optimizing aerosolization efciency of dry-powder aggregates of thermally-sensitive polymeric nanoparticles produced by spray-freeze-drying
Katherine Kho, Kunn Hadinoto
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore

a r t i c l e

i n f o

a b s t r a c t
Dry powder inhaler (DPI) delivery of therapeutic nanoparticles requires the nanoparticles to be transformed into inhalable micro-scale aggregate structures (i.e. nano-aggregates). The present work details the sprayfreeze-drying (SFD) production of dry-powder aggregates of thermally-sensitive polymeric nanoparticles. Specically, the aim is to optimize the aerosolization efciency of the nano-aggregates, while keeping the morphology, production yield, owability, and aqueous reconstitution in the desirable range. For this purpose, the effects of SFD process parameters (i.e. atomization rate, feed concentration, and feed rate) and freeze-drying adjuvant formulation on the nano-aggregate characteristics are examined. Low melting-point poly (caprolactone) (PCL) nanoparticles are used as the model nanoparticles. Mannitol and leucine are used as the hydrophilic and hydrophobic adjuvants, respectively. Large spherical porous nano-aggregates, where PCL nanoparticles are physically dispersed in the porous adjuvant matrix, have been produced. The presence of mannitol is crucial in ensuring that the nano-aggregates readily reconstitute into individual nanoparticles upon exposure to an aqueous environment, so that they can perform their intended therapeutic functions. The presence of leucine, on the other hand, is mandatory to obtain high aerosolization efciency as its presence reduces the nano-aggregate tendency to agglomerate. At the optimal condition, nano-aggregates exhibiting ED (Emitted Dose) 95%, FPF (Fine Particle Fraction) 30%, and MMAD (Mass Median Aerodynamic Diameter) 5.3 m, which are comparable to the values obtained in commercial DPI, have been produced. The results signify the potential of SFD to be employed in the production of inhalable dosage form of thermally-sensitive therapeutic nanoparticles. 2011 Elsevier B.V. All rights reserved.

Article history: Received 17 March 2011 Received in revised form 4 August 2011 Accepted 9 August 2011 Available online 16 August 2011 Keywords: Nanoparticles Spray freeze drying Dry powder inhaler Aerosols Aggregates

1. Introduction The use of nanoparticles as drug delivery vehicles in the treatment of pulmonary diseases has gained signicant interests as the nanoscale formulation enables the therapeutic agents to evade the lung phagocytic clearance mechanism and enhances their bioavailability in the lung through targeted delivery [1]. As an inhaled nanoparticle delivery platform, dry powder inhaler (DPI) offers numerous advantages, such as portability and longer shelf-life, over the more conventional delivery platform by nebulization of aqueous suspension of the nanoparticles. In inhaled drug delivery, aerodynamic diameter (da), which is a function of the geometric diameter (dg) and particle effective density (eff) (Eq. 1), is used to characterize the particle deposition tendency in the human respiratory airways. s eff 3 where s = 1 g=cm da = dg s

Corresponding author. Tel.: + 65 6514 8381; fax: + 65 6794 7553. E-mail address: kunnong@ntu.edu.sg (K. Hadinoto). 0032-5910/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2011.08.010

In order to effectively deposit in the lung, particles must possess da between 1 and 5 m [2]. Particles with da smaller than 1 m tend to be exhaled, whereas particles with da larger than 10 m tend to deposit in the mouth and throat regions. The small dg of nanoparticles results in da 1 m, such that nanoparticles delivered by inhalation are predominantly exhaled. In addition, dry-powder nanoparticles have a strong tendency to agglomerate due to the small dg resulting in poor aerosolization and difcult physical handling. To facilitate their effective delivery to the lung by DPI, nanoparticles are typically transformed into their micro-scale aggregated form having large dg (N 10 m) and low eff (b 1 g/cm 3) [3]. The low eff, which is attributed to either porous or hollow morphology, compensates for the large dg resulting in da within the 15 m range. The most common technique employed to produce the large and low-density nanoparticle aggregates (i.e. nano-aggregates) is by spray drying (SD). By manipulating the spray-drying conditions and the drying adjuvant formulation, dry-powder aggregates of various nanoparticle types (e.g. polymers, silica) exhibiting high aerosolization efciency have been successfully produced [3,4]. In addition to the effective aerosolization, the spray-dried nano-aggregates have been specically formulated, by water-soluble adjuvant inclusion, to

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readily reconstitute into individual nanoparticles in an aqueous environment, such as the lung interstitial uid, so that they can perform their intended therapeutic functions. The SD technique, however, necessitates the use of high temperatures ( 100 C) to achieve a fast convective drying rate required to obtain the low eff [5]. Consequently, the SD technique is not suitable for producing nano-aggregates of thermally-sensitive drugs and their carriers (e.g. polymers, liposomes). Our previous work on spray drying of thermally-sensitive poly(caprolactone) (PCL) nanoparticles [6] showed that even though large hollow nano-aggregates were successfully produced with the help of drying adjuvants, the physical integrity of the PCL nanoparticles was compromised due to exposures to temperatures high above the melting point, which resulted in irreversible nanoparticle coalescences that diminished their therapeutic functions. In this regard, spray-freeze-drying (SFD) offers a distinct advantage over SD by eliminating the need for high-temperature operations. The SFD technique has been widely employed in the dry-powder preparation of thermally-labile proteins [7]. In the SFD process, atomized droplets of the nanoparticle suspension are rapidly frozen in liquid nitrogen, followed by lyophilization of the ice crystals to produce drypowder micro-scale aggregates of the nanoparticles. Particles produced by SFD typically possess larger dg than those produced by SD, because the rapid freezing step, followed by sublimation in SFD, results in particle size that is comparable to the initial droplet size, unlike in SD that relies on droplet evaporation hence shrank particle size. In addition, SFD particles are inherently porous yielding low eff owed to the interstitial sublimation of the ice crystals in the frozen droplets. As a result, the SFD technique is highly suitable to produce nano-aggregates having large dg and low eff. Our rst attempt on studying SFD of PCL nanoparticles in Cheow et al. [8] demonstrated that large porous aggregates of PCL nanoparticles, having the desired dg ( 2040 m) and the desired theoretical da ( 35 m) calculated from Eq. (1), were successfully produced. The effects of freeze-drying adjuvant (only water-soluble ones) formulation on the morphology (i.e. dg, eff, and da), production yield, and aqueous reconstitution of the nano-aggregates were investigated. Inclusion of a large amount of hydrophilic adjuvants, such as mannitol at 70% (w/w), or polyvinyl alcohol (PVA) at 40% (w/w), was found necessary to produce nano-aggregates that could reconstitute into individual nanoparticles upon exposure to aqueous media. The role of the adjuvants here is to form interparticle bridges that prevent nanoparticles from being in direct contact with each other during lyophilization. Thereby, upon dissolution of the adjuvant bridges in aqueous media, the nano-aggregates readily reconstitute into individual nanoparticles, though the extent of reconstitution may vary depending on the adjuvant formulation. Despite having the desired morphology and reasonable aqueous reconstitutibility, the nano-aggregates produced in Cheow et al. [8] exhibit low ne particle fractions (FPF) (i.e. 10%), which suggests poor particle deposition efciency in the lung. The low FPF, which is dened as the fraction of particles with da 5 m, is caused by agglomeration of the nano-aggregate powders upon aerosolization. The agglomeration is due to their cohesive nature attributed to the moisture uptake of the hydrophilic adjuvants. To follow up the work of Cheow et al. [8], herein we aim to improve the aerosolization efciency of the nano-aggregates by reducing their degree of cohesiveness, while maintaining the particle morphology, owability, aqueous reconstitutibility, and production yield in the optimal range. In this regard, the adjuvant formulation and SFD process parameters (e.g. atomization rate, feed concentration) work hand-in-hand in inuencing the nano-aggregate characteristics, therefore the effects of both must be investigated concurrently. In the rst approach, SFD experiments employing a smaller amount of hydrophilic adjuvant than that used in Cheow et al. [8] to reduce the degree of cohesiveness will be performed. The effect of SFD process parameters on the nano-aggregate characteristics, which were not

investigated in Cheow et al. [8], at a lower hydrophilic adjuvant amount will be studied. In the second approach, besides reducing the hydrophilic adjuvant amount, inclusion of hydrophobic adjuvants is thought to be able to further reduce the degree of cohesiveness. The presence of hydrophobic adjuvants, however, may negatively inuence the other nano-aggregate characteristics, particularly the aqueous reconstitution. Therefore, the effects of both SFD process parameters and hydrophilichydrophobic adjuvant formulation will be concurrently examined. Low melting point PCL ( 62 C) is used as the thermally-sensitive polymeric nanoparticle model. PCL has been widely investigated as drug carriers due to its biodegradability, non-toxicity, and high matrix permeability towards a wide range of drugs [9]. Levooxacin antibiotic is loaded into the PCL nanoparticles to create a model for inhalable antibiotic nanoparticles to treat respiratory infections. Mannitol and leucine, which both have acquired generally-recognized-as-safe (GRAS) status, are used as the hydrophilic and hydrophobic adjuvants, respectively. Mannitol is used as the hydrophilic adjuvant, instead of PVA, because inhaled mannitol has been found to improve mucus clearance in infected lungs [10]. 2. Materials and methods 2.1. Materials PCL (MW = 80,000), levooxacin (LEV), polyvinyl alcohol (PVA, MW = 23,000), dichloromethane (DCM) are used in the nanoparticle preparation. D-mannitol and L-leucine are used in the SFD process. Sodium periodate, sodium thiosulfate, acetylacetone, and ammonium acetate are used in the mannitol colorimetric assay. All chemicals used are purchased from Sigma-Aldrich (USA). 2.2. Methods 2.2.1. PCL nanoparticle preparation and characterization LEV-loaded PCL nanoparticles are prepared using an emulsicationsolvent-evaporation method. Briey, 8 mg of LEV and 80 mg of PCL are dissolved in 2 mL of DCM as the organic phase, which is then poured into 6 mL of 1.0% (w/v) aqueous PVA solution. The resultant mixture is emulsied for 1 min using a Vibra-Cell probe sonicator (Sonics & Materials, USA). The resultant nano-emulsion is transferred into 10 mL of 0.1% (w/v) aqueous PVA solution and stirred overnight at room temperature to evaporate off the DCM resulting in the production of PCL nanoparticles. Afterwards, the nanoparticle suspension is centrifuged twice at 13,000 rpm for 12 min to remove the residual PVA and LEV. The original nanoparticle size (Si) is measured using a Brookhaven 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments Corporation, USA). 2.2.2. Spray-freeze-drying production of dry-powder aggregates of PCL nanoparticles The feed suspension is prepared by mixing PCL nanoparticle suspension with aqueous solution of the adjuvants. The SFD process is performed in a modied BCHI B-290 mini spray-dryer (BCHI, Switzerland) illustrated in Fig. 1, where the drying chamber of the spray dryer is removed and replaced with a polypropylene vessel containing 400 mL of liquid nitrogen under constant stirring at 500 rpm. A two-uid atomizer with 1.5 mm nozzle diameter is used because it offers longer operating time with minimal variations than ultrasonic atomizers, which are prone to temperature rise that may affect the SFD process. The distance between the nozzle tip and the liquid nitrogen surface is xed at 10 cm. After the spray-freezing step, the slurry containing the frozen droplets is transferred to a lyophilization container by rst evaporating the excess liquid nitrogen. The frozen droplets are then lyophilized at 52 C and 0.05 mbar for 24 h in Alpha 12 LD Plus freeze dryer (Martin Christ, Germany).

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2.2.3.2. Aqueous reconstitution. Briey, 10 mg of the nano-aggregates is added into 2 mL of deionized water under gentle stirring. The nanoparticle size after reconstitution (Sf) is measured in triplicates. The ratio of Sf/Si indicates the extent of reconstitution, where fully reconstituted nano-aggregates exhibit Sf/Si ratio 1 [12]. To determine the fraction of the nano-aggregates that have reconstituted (i.e. % reconstitution), the suspension is centrifuged at 6000 rpm for 10 min after which 1.5 mL of the supernatant is discarded to remove the reconstituted nanoparticles and the dissolved adjuvants. The supernatant is replaced with 1.5 mL of deionized water and the centrifugation step is repeated three times. At the end of the fourth centrifugation, after 1.5 mL of the supernatant is discarded, the sedimented pellet is lyophilized for 24 h. The lyophilized pellet is weighted and the % reconstitution is calculated with respect to the initial amount of nano-aggregates supplied. 2.2.3.3. Aerosolization efciency. The aerosolization efciency is characterized using a seven-stage Next Generation Pharmaceutical Impactor (NGI; Copley Scientic, UK) equipped with an induction port (IP) and a pre-separator (PS). The aerosolization efciency is characterized using two replicates. Four parameters are used to examine the aerosolization efciency, i.e. emitted dose (ED), ne particle fraction (FPF), mass median aerodynamic diameter (MMAD), and cohesion index. ED is dened as the amount of powders successfully aerosolized off the inhaler expressed as a percentage of the total amount of powders initially placed in the inhaler. FPF is dened as the amount of powders with da 5 m expressed as a percentage of the total amount of powders collected in the impactor. MMAD is dened as the median da value determined from the cumulative mass distribution plot of da. The cohesion index, which characterizes the degree of nano-aggregate agglomeration upon aerosolization, is determined from the ratio of MMAD to the theoretical da (Eq. 1), where high cohesion index indicates strong agglomeration upon aerosolization [13]. A modied powder entrainment tube (PET) based on the work of Louey et.al. (2006) [14] is employed in place of an inhaler. Aerosolization performance of the PET has been demonstrated to be comparable to that of two commercial inhalers i.e. inhalator and rotahaler [14]. The use of PET enables evaluations of the aerosolization efciency to be independent of the type of inhaler used. Furthermore, the PET eliminates the dosing complexities of commercial inhalers, such as the need to use capsules and blisters. The PET consists of three sections namely inlet, powder dosage area, and outlet (Fig. 2). A fully-developed turbulent airow is generated in the inlet to entrain 5 mg of particles placed in the dosage area. A removable mesh is included in the powder dosage area to assist powder dispersions. The outlet of the PET is connected to IP of the NGI. The airow rate needed to achieve the recommended 4 kPa pressure drop over the PET is 85 L/min determined using critical ow controller (Copley Scientic, UK). The effective cut-off da for each stage at 85 L/min is 6.7, 3.7, 2.4, 1.4, 0.8, 0.5 and 0.3 m from stages 1 to 7, respectively. The ow duration is set to 2.8 s to draw 4 L of air that simulates typical human inhalation volume. The PS and impactor stages are coated with silicone grease to prevent particle re-entrainment after deposition. Powders recovered in the IP, PS, and impactor stages are quantied using a mannitol colorimetric assay [15]. From the amount of mannitol, the amount of powders recovered in each stage is determined from the known nanoparticle to adjuvant ratio. Briey, 2 mL aliquot of the aqueous suspension of the recovered powders is centrifuged at 14,000 rpm for 12 min. The supernatant is collected and diluted to 2 mL with deionized water. 1 mL of 5 mM sodium periodate is added to the diluted supernatant and is agitated for 15 s. Afterwards, 1 mL solution containing 0.1 M acetylacetone, 2 M ammonium acetate, and 0.02 M sodium thiosulfate is added and agitated for 15 s. The mixture is heated at 100 C for 2 min followed by cooling in an ice bath. The absorbance is measured at 412 nm

Fig. 1. Schematic diagram of the spray-freeze-drying (SFD) process.

2.2.3. Characterizations of dry-powder aggregates of PCL nanoparticles 2.2.3.1. Morphology, production yield, and owability. The nanoaggregate morphology is characterized using scanning electron microscope (SEM) model JSM-6700 F (JEOL, USA). The volumeaveraged dg is determined by measuring triplicates of 1000 particles from the SEM images using ImageJ software (NIH, USA). The image analysis method is preferred over the conventional laser diffraction method that employs mechanical agitation and sonication prior to the measurement to disperse the particles, which leads to the nanoaggregates being reconstituted prematurely, hence resulting in underestimated dg values. eff is determined from tap density (tap) using a tap densitometer (Quantachromme, USA) based on 2000 taps of 1 mL powder in two replicates. The theoretical da value is calculated from Eq. (1) using the measured values of dg and eff. The production yield (i.e. Yield) is determined from the ratio of nano-aggregate mass collected to the initial mass in the feed suspension (i.e. PCL nanoparticles + adjuvants). The owability is characterized by Carr's compressibility index (CI) calculated from Eq. (2), where CI values below 25 indicate good owability and CI values above 40 indicate poor owability [11]. The bulk density (bulk) is determined by measuring the volume of powders of a known mass in a 5 mL measuring cylinder without tapping. Yield and CI measurements are performed in two replicates. CI = 1 bulk tap ! 100% 2

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Fig. 2. Powder entrainment tube (PET) for the aerosolization efciency study (all dimensions are in mm).

wavelengths with UVVis spectrophotometer (UV Mini-1240, Shimadzu, Japan). 3. Results and discussion 3.1. First approach by lowering the hydrophilic adjuvant amount The ratio of PCL nanoparticles to mannitol (i.e. P/M ratio) is xed at 1:1 (i.e. 50% w/w mannitol), which is higher than the 3:7 ratio (i.e. 70% w/w mannitol) used in Cheow et al. [8]. P/M ratios higher than 1:1 lead to poor aqueous reconstitution of the nano-aggregates therefore they are not pursued here. At this lower mannitol amount, the effects of three SFD process parameters i.e. total solid concentration (TS), feed rate (FR), and atomization rate (AR) on dg, eff, da, Yield, CI, and Sf/Si on the nanoaggregate characteristics are investigated by 23 factorial designs. The results are presented in Table 1 as Runs A1A8. The experimental uncertainties in the values of dg, da, Yield, CI, and Sf/Si are 7%, 8%, 10%, 7%, and 3%, respectively. The TS, FR, and AR values investigated range from 2.5 to 4.0% (w/v), 0.13 to 0.27 L/h, and 250 to 350 L/h, respectively. For comparison, Cheow et al. [8] performed their SFD experiment at xed FR= 0.24 L/h and AR = 240 L/h. 3.1.1. Effects of SFD process parameters on morphology, production yield, and owability Two runs conducted at the lower limits of both FR and AR (i.e. Runs A1 and A5) produce highly fragile nano-aggregates with signicant amount of fragments, hence the results are not reported here. For runs that produce structurally robust nano-aggregates (i.e. Runs A2A4 and A6A8), the particles produced exhibit large dg and spherical shape as shown in Fig. 3A. A close-up look at the nano-aggregate surface in Fig. 3B

reveals that PCL nanoparticles, whose size (Si) is 260 10 nm, are physically dispersed in the porous mannitol matrix. The failed Runs A1 and A5 prevent us from analyzing the factorial design experimental results to identify the statistically signicant parameters. As an alternative, the effects of FR and AR on dg, eff, and da are examined separately in Fig. 4A and B, respectively, as a function of TS. The da lines in Fig. 4 represent the theoretical da values calculated from Eq. (1) for given values of dg and eff. The theoretical da lines serve a purpose of

Table 1 A summary of PCLmannitol nano-aggregate characteristics. Run A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 TS (% w/v) 2.5 2.5 2.5 2.5 4.0 4.0 4.0 4.0 5.5 7.0 FR (L/h) 0.13 0.13 0.27 0.27 0.13 0.13 0.27 0.27 0.27 0.27 AR (L/h) 250 350 250 350 250 350 250 350 410 440 Yield CI Sf/Si

Fragile aggregates 21% 40 16% 40 29% 24 Fragile aggregates 35% 27 30% 31 37% 26 52% 15 48% 16

2.9 2.9 2.8 2.0 2.2 1.3 2.1 1.7

Fig. 3. (A) PCLmannitol nano-aggregates exhibit large size and spherical shape; (B) PCL nanoparticles are physically dispersed in the porous mannitol matrix.

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identifying the ranges of dg and eff that yield the desired da range ( 1 5 m). First, the effects of FR on dg and da are examined at a constant AR = 350 L/h in Fig. 4A. At TS= 2.5% (w/v) (i.e. Runs A2 and A4), the effects of FR variation on dg and da are minimal, where particles having dg 20 m and da 23 m are produced. Similar observations are made at TS= 4.0% (w/v) (i.e. Runs A6 and A8) though the particles have slightly larger da ( 34 m). The effect of FR on the production yield is also relatively insignicant, where on average the yields are equal to 25% and 35% at TS= 2.5 and 4.0% (w/v), respectively. The yield can therefore be increased by increasing TS without any signicant impacts on dg and da. Second, the effects of AR on dg and da are examined at a constant FR = 0.27 L/h in Fig. 4B. At TS = 2.5% (w/v) (i.e. Runs A3A4), dg of the particles are signicantly reduced from 50 m to 20 m when AR is raised from 250 to 350 L/h, which is not unexpected as higher AR at constant FR results in smaller droplets. The eff values of the particles, however, remain constant at 0.02 g/cm 3 despite the dg variation.

Consequently, da of particles in Run A3 (N 5 m) is signicantly larger than that of Run A4 ( 3 m). A similar trend in the dg variation as a function of AR is observed at TS = 4.0% (w/v) (i.e. Runs A7A8). Unlike Runs A3A4, however, eff of Run A8 is considerably higher than that of Run A7. Importantly, the results of Runs A3 and A7 reafrm the earlier nding that the production yield can be increased by increasing TS with minimal impacts on dg and da. On this note, due to their theoretical da values being larger than 5 m, Runs A3 and A7 are excluded from subsequent discussions. In terms of the owability, Runs A4, A6, and A8 exhibit CI values 25 2 signifying good powder owability. The high CI value in Run A2 ( 40) is best interpreted as an experimental outlier, possibly due to sample preparation error, because the particles in Run A2 possess nearly identical dg and eff as those in Runs A4 and A6, such that they should have exhibited similar owability, everything else being equal. Lastly, the geometric particle size distributions in Fig. 5A, represented by that of Run A8, denotes a rather wide distribution between 20 and 60 m, which is an inherent characteristic of SFD powders due to the wide size distribution of the sprayed droplets. 3.1.2. Subsequent impacts on aqueous reconstitution In terms of the aqueous reconstitution, only Run A8 exhibits reasonable Sf/Si 1.3, whereas the other three runs (i.e. Runs A2, A4, and A6) exhibit Sf/Si 2.0. The lower Sf/Si in Run A8, which possesses similar dg, but higher eff compared to the other three runs, suggests the dependence of aqueous reconstitution on eff. Importantly, the low Sf/Si of Run A8 obtained at P/M ratio = 1:1 is comparable to that obtained in Cheow et al. [8] at P/M ratio = 3:7. The % reconstitution of Run A8 at 60 5%, however, is lower than 85% obtained in Cheow et al. [8]. Due to the higher P/M ratio used here. To further investigate the effect of eff on Sf/Si, two additional runs (i.e. Runs A9A10) are performed at higher TS (i.e. 5.5 and 7.0% w/v) aimed to increase eff, while keeping FR at 0.27 L/h. To maintain da 15 m, AR is simultaneously increased with TS in Runs A9A10 in order to prevent dg from getting too large. The results in Fig. 5B indicate that higher eff are indeed obtained, however, with smaller dg ( 10 m) resulting in slightly smaller da ( 23 m). Higher production yields up to 52% are also obtained, which is not unexpected as TS is increased. The higher eff in Runs A9 A10 ( 0.040.045 g/cm 3) also enhances the owability (CI 15) despite the smaller dg. The higher eff, however, does not lead to improvement in Sf/Si, where the values in fact increase to 2.1 and 1.7 for Runs A9 and A10, respectively, indicating poorer aqueous reconstitution. The results hence indicate that both dg and eff are important in obtaining the desired Sf/Si values. 3.1.3. Subsequent impacts on aerosolization efciency All particles produced in Runs A2, A4, A6, A8, and A9A10 exhibit similar values of FPF and MMAD, where they exhibit low FPF ( 10%) with MMAD 8.0 m similar to the ndings of Cheow et al. [8], despite the 20% reduction in the mannitol content. Their ED values, nevertheless, range from 75 to 90%, which are comparable to commercial inhalation products. The high ED values indicate that entrainment of these particles off the PET is not an issue. Instead, the poor FPF and MMAD are caused by the nano-aggregates forming agglomerates airborne, which are difcult to disperse, resulting in a signicant fraction of the particles ( 65%) recovered in the IP and PS of the NGI (Fig. 6). In summary, SFD at a lower mannitol content (i.e. higher P/M ratio) can produce highly reconstitutable nano-aggregates having the desired dg and theoretical da values, as well as good owability and production yield, provided that the SFD process parameters, particularly TS and AR, are optimized, as exemplied by Run A8. However, the lower mannitol content, does not lead to any improvements in the aerosolization efciency. As the P/M ratio cannot be increased further, the effects of multiple excipient formulations involving both hydrophilic and hydrophobic adjuvants are investigated next.

Fig. 4. Effects of (A) feed rate (FR) and (B) atomization rate (AR) on dg, eff, and da at two different total solid concentrations (TS).

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A
30 A8

% volume

aggregates, which would render the particles less cohesive hence improving the aerosolization efciency. Furthermore, the presence of leucine on particle surfaces has also been found to lower the surface free energy therefore reducing the particle tendency to agglomerate [16]. In addition, leucine inclusion is known to produce particles with corrugated surfaces upon drying, which reduce interparticle contact surface areas, resulting in better particle dispersions [13]. 3.2.1. Effects on morphology, production yield, owability, and aqueous reconstitution The effects of incorporating leucine into the PCLMannitol formulation on dg, eff, da, Yield, CI, and Sf/Si are investigated in Runs B1B6 presented in Table 2. The investigations are performed at constant FR = 0.27 L/h as the effects of FR have been found earlier to be insignicant. In Runs B1B6, the effects of varying the mannitol to leucine concentration ratio (i.e. M/L ratio) at different TS are investigated for M/L ratios of 6:1 and 5:2. The PCL nanoparticle concentration is xed at one-eighth of TS because higher nanoparticle concentrations would require even higher M/L ratios to reconstitute the nano-aggregates. The high M/L ratio is required because spray-freeze-dried leucine particles are highly fragile [8], such that when leucine is included in the formulation to improve the aerosolization efciency, a larger amount of mannitol is needed to maintain the structural integrity of the nano-aggregates. In addition, the presence of leucine decreases the aqueous reconstitution due to the low aqueous solubility of leucine ( 25 mg/mL) compared to 180 mg/mL for mannitol. Consequently, a larger amount of mannitol is needed for reconstitution to counteract the presence of leucine. Starting at TS= 4.0% (w/v), which is found earlier to be optimal in the PCLMannitol formulation, spherical particles (Fig. 7A), with dg 14 m and eff 0.02 g/cm 3 resulting in da 2 m, are successfully produced at AR= 410 L/h (i.e. Runs B1B2). Because of the larger presence of mannitol, which tends to produce large particles, higher AR is needed to maintain dg within the 1030 m range, so that particles with theoretical da 15 m can be obtained. A close-up view of the PCLmannitolleucine nano-aggregates in Fig. 7B denotes a highly corrugated surface attributed to the leucine inclusion. Not unexpectedly, the CI values ( 34) indicate reasonably good owability owed to the corrugated surface, despite the high mannitol content. The production yield, however, is rather low at 20%. The effects of M/L ratio in Runs B1B2 on dg, eff, and da (Fig. 8), as well as on the Yield and CI, are found to be minimal. The higher M/L ratio in Run B1 nonetheless results in lower Sf/Si compared to Run B2 (i.e. 1.2 versus 1.9) denoting reasonable reconstitutibility. To increase the production yield, TS is increased to 5.5% (w/v) at AR = 440 L/h in Runs B3B4. The production yield is increased to 4050% as expected. The effects of increasing TS from 4.0 to 5.5% (w/v) on dg, da, and CI are fairly insignicant, though not negligible. Particles having considerably higher eff ( 0.04 g/cm 3) are produced (Fig. 8). Moreover, the effect of M/L ratio on Sf/Si is diminished, where both M/L ratios result in Sf/Si 1.3 denoting good reconstitutibility with % reconstitutions above 60%. In contrast, the effect of M/L ratio on dg is intensied, where the lower M/L ratio produces considerably

20

10

0 0 20 40 60 80

dg (m)

B
0.05
A9

0.04

A10

FR = 0.27 L/h

eff (g/cm3)

0.03

A8

0.02
2 m 3 m 4 m 5 m

0.01
da = 1.5 m

0 5 15 25 35 45 55

dg (m)
Fig. 5. (A) Geometric size distribution of Run A8 particles; (B) Effects of TS and AR variations on dg, eff, and da.

3.2. Second approach by inclusion of both hydrophilic and hydrophobic adjuvants Owing to the hydrophobic nature of leucine, its inclusion is anticipated to limit the moisture uptake of the PCLMannitol nano-

Table 2 A summary of PCLmannitolleucine nano-aggregate characteristics. Run B1 B2 B3 B4 B5 B6 TS (% w/v) 4.0 5.5 2.4 PCL (% w/v) 0.5% 0.7% 0.3% AR (L/h) 410 440 350 M/L 6:1 5:2 6:1 5:2 6:1 5:2 Yield 20% 21% 37% 49% 36% 35% CI 34 34 31 33 30 22 Sf/Si 1.2 1.9 1.3 1.3 1.8 1.8

Fig. 6. Typical particle deposition patterns of PCLmannitol nano-aggregates.

K. Kho, K. Hadinoto / Powder Technology 214 (2011) 169176 Table 3 Aerosolization characteristics of PCLmannitolleucine nano-aggregates. Run ED FPF ( 5 m) MMAD Cohesion index B1 88% 22% 6.5 3.4 B2 92% 32% 5.3 2.9 B3 92% 20% 7.2 2.5 B4 96% 30% 5.5 3.4 B5 94% 17% 7.8 3.7

175

B6 95% 29% 5.3 2.0

(Fig. 8). The nano-aggregates are poorly reconstituted as manifested in the high Sf/Si values ( 1.8), regardless of the M/L ratios. The effects of M/L ratio on dg, eff, Yield, and CI are also insignicant. The production yield nonetheless is unexpectedly higher than that obtained at TS = 4.0% (w/v). Taking into account the particle morphology, production yield, owability, and aqueous reconstitution, Runs B3B4 are determined as the optimal SFD run for the PCL MannitolLeucine formulation. 3.2.2. Effects on aerosolization efciency The ED, FPF, MMAD, and cohesion index values of the particles produced in Runs B1B6 are summarized in Table 3. The experimental uncertainties in ED, FPF, MMAD, and cohesion index based on two replicates are 8%, 3%, 3%, and 11%, respectively. All six runs demonstrate ED 90% that signies highly effective particle entrainment off the PET, which was also observed with the PCLmannitol nanoaggregates. Signicantly, the leucine inclusion enhances the FPF and MMAD as postulated. Runs with M/L ratio= 5:2 (i.e. Runs B2, B4, and B6) exhibit higher FPF and lower MMAD values compared to those of Runs with M/L ratio= 6:1 (i.e. Runs B3, B5, and B7). Therefore, the higher the leucine content, the higher (lower) the FPF (MMAD). The representative particle deposition patterns of Runs B2, B4, and B6 are provided in Fig. 9. A signicant fraction of particles are recovered in stages 24 representing vast improvement over the PCLmannitol formulation presented in Fig. 6. The cohesion index, however, remains high ranging from 2.0 to 3.7 denoting still signicant agglomeration of the nano-aggregates, despite the leucine presence. Notably, runs with the same M/L ratio exhibit similar FPF and MMAD values irrespective of their dg and eff values, which signify that the particle morphology has less impact towards aerosolization efciency than the adjuvant formulation. Taking all factors into consideration, Run B4 clearly represents the optimal SFD process parameters and adjuvant formulation, where Yield 50%, Sf/Si 1.3, ED 96%, and FPF 30% are obtained. The high FPF and low MMAD, which are comparable to the values exhibited by commercial DPI, are undeniably obtained at the expense of having higher adjuvant to nanoparticle ratios, particularly of mannitol.

Fig. 7. (A) PCLmannitolleucine nano-aggregates exhibit large size and spherical shape; (B) their corrugated surface due to leucine inclusion.

smaller dg. The effects of M/L ratio on the rest of the parameters remain relatively insignicant. In terms of the particle size distribution, the nano-aggregates produced in Runs B3B4 exhibit similar size distributions as that presented in Fig. 5A, hence they are not presented here for brevity. For comparison, at a lower TS = 2.4% (w/v) and AR = 350 L/h in Runs B5B6, slightly larger particles having lower eff are produced

0.04
B4

B3

0.03

eff (g/cm3)

0.02

B1 B2 da= 1.5 m 2 m B5 3 m B6 4 m

0.01

0 5 15 25 35
Fig. 9. Typical particle deposition patterns of PCLmannitolleucine nano-aggregates in Runs B2, B4, and B6.

dg (m)
Fig. 8. Effects of different PCLmannitolleucine formulations on dg, eff, and da.

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However, it has to be pointed out that, unlike the conventional DPI delivery, these nano-aggregates are readily aerosolized without the assistance of coarse carrier particles (e.g. lactose) owed to their unique morphology (i.e. large dg and low eff). As a result, the amounts of adjuvants used exclusively for delivery purposes have been essentially reduced compared to the conventional delivery mode. From that perspective, the carrier-free aerosolization here can adequately justify for the high adjuvant to nanoparticle ratio required. 4. Conclusion Two studies aimed to improve the aerosolization efciency of spray-freeze-dried aggregates of thermally-sensitive polymeric nanoparticles have been performed via optimizations of both the SFD process parameters and adjuvant formulations. In addition to the aerosolization efciency, the optimization also takes into account the particle morphology, production yield, owability, and aqueous reconstitution. In the rst study, the hydrophilic adjuvant content (i.e. mannitol) is lowered to the minimal amount below which the nano-aggregates cannot sufciently reconstitute aimed to reduce particle cohesiveness. Poor aerosolization efciency as manifested by the low ne particle fractions ( 10%), however, remains observed independent of the other nano-aggregate characteristics. In the second study, the inclusion of both hydrophilic (i.e. mannitol) and hydrophobic (i.e. leucine) adjuvants signicantly improves the ne particle fraction ( 30%), without jeopardizing the other nanoaggregate characteristics, as long as the SFD process parameters and mannitol to leucine ratio are optimized. The particle size distribution, however, still leaves much room for improvement in terms of the monodispersity. Experimental works on improving the particle size distribution by controlling the sprayed droplet size are currently ongoing in our laboratory. Nomenclature AR atomization ow rate (L/h) da aerodynamic diameter (m) dg geometric diameter (m) ED emitted dose (% w/w) FPF ne particle fraction (% w/w) FR feed rate (L/h) MMAD mass median aerodynamic diameter (m) M/P mannitol to PCL concentration ratio (% w/w) M/L mannitol to leucine concentration ratio (% w/w) eff particle effective density (g/cm 3) s particle unit density (1 g/cm 3) tap particle tap density (g/cm 3) bulk particle bulk density (g/cm 3) CI Carr's index Si raw nanoparticle size (nm) Sf nanoparticle size after aqueous reconstitution (nm)

Sf/Si TS Yield

ratio of raw to reconstituted nanoparticle size total solid concentration (% w/v) nano-aggregate production yield (%)

Acknowledgment The authors would like to thank Jellynn Teng and Prima Dewi Sinawang for their contributions in the aerosolization efciency characterization study. Financial supports from Nanyang Technological University's Start-Up Grant (SUG 8/07) and Undergraduate Research Experience on Campus (URECA) are gratefully acknowledged. References
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