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Optimizing aerosolization efficiency of dry-powder aggregates of thermally-sensitivepolymeric nanoparticles produced by spray-freeze-drying

Katherine Kho, Kunn Hadinoto

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore

a b s t r a c ta r t i c l e i n f o

Article history:

Received 17 March 2011

Received in revised form 4 August 2011Accepted 9 August 2011Available online 16 August 2011

Keywords:

NanoparticlesSpray freeze dryingDry powder inhalerAerosolsAggregates

Dry powder inhaler (DPI) delivery of therapeutic nanoparticles requires the nanoparticles to be transformedinto inhalable micro-scale aggregate structures (i.e. nano-aggregates). The present work details the spray-freeze-drying (SFD) production of dry-powder aggregates of thermally-sensitive polymeric nanoparticles.Specifically, the aim is to optimize the aerosolization efficiency of the nano-aggregates, while keeping themorphology, production yield, flowability, and aqueous reconstitution in the desirable range. For thispurpose, the effects of SFD process parameters (i.e. atomization rate, feed concentration, and feed rate) andfreeze-drying adjuvant formulation on the nano-aggregate characteristics are examined. Low melting-pointpoly(caprolactone) (PCL) nanoparticles are usedas the model nanoparticles. Mannitol and leucine are usedasthe hydrophilic and hydrophobic adjuvants, respectively. Large spherical porous nano-aggregates, where PCLnanoparticles are physically dispersed in the porous adjuvant matrix, have been produced. The presence ofmannitol is crucial in ensuring that the nano-aggregates readily reconstitute into individual nanoparticlesuponexposureto an aqueous environment, so that they can perform their intendedtherapeutic functions. Thepresence of leucine, on the other hand, is mandatory to obtain high aerosolization efficiency as its presencereduces the nano-aggregate tendency to agglomerate. At the optimal condition, nano-aggregates exhibitingED (Emitted Dose)95%, FPF (Fine Particle Fraction)30%, and MMAD (Mass Median AerodynamicDiameter)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.

1. Introduction

The use of nanoparticles as drug delivery vehicles in the treatmentof pulmonary diseases has gained significant interests as thenanoscale formulation enables the therapeutic agents to evade thelung phagocytic clearance mechanism and enhances their bioavail-ability in the lung through targeted delivery [1]. As an inhalednanoparticle delivery platform, dry powder inhaler (DPI) offersnumerous advantages, such as portability and longer shelf-life, overthe more conventional delivery platform by nebulization of aqueoussuspension of the nanoparticles. In inhaled drug delivery, aerody-namic diameter (da), which is a function of the geometric diameter(dg) and particle effective density (eff) (Eq. 1), is used to characterizethe particle deposition tendency in the human respiratory airways.

da = dg

ffiffiffiffiffiffiffiffieff

s

swhere s = 1 g=cm

31

In order to effectively deposit in the lung, particles must possessda between1and5 m [2]. Particleswith da smaller than 1 mtendtobe exhaled, whereas particles with da larger than 10 m tend todeposit in the mouth and throat regions. The small d g of nanopar-ticles results in da1 m, such that nanoparticles delivered byinhalation are predominantly exhaled. In addition, dry-powdernanoparticles have a strong tendency to agglomerate due to thesmall dg resulting in poor aerosolization and difficult physicalhandling. To facilitate their effective delivery to the lung by DPI,nanoparticles are typically transformed into their micro-scaleaggregated form having large dg (N10 m) and low eff (b1 g/cm

3)[3]. The low eff, which is attributed to either porous or hollowmorphology, compensates for the large dg resulting in da within the15 m range.

The most common technique employed to produce the large andlow-density nanoparticle aggregates (i.e. nano-aggregates) is by spraydrying (SD). By manipulating the spray-drying conditions and thedrying adjuvant formulation, dry-powder aggregates of variousnanoparticle types (e.g. polymers, silica) exhibiting high aerosoliza-tion efficiency have been successfully produced [3,4]. In addition tothe effective aerosolization, the spray-dried nano-aggregates havebeen specifically formulated, by water-soluble adjuvant inclusion, to

Powder Technology 214 (2011) 169176

Corresponding author. Tel.: +65 6514 8381; fax: +65 6794 7553.E-mail address: [email protected] (K. Hadinoto).

0032-5910/$ see front matter 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.powtec.2011.08.010

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http://dx.doi.org/10.1016/j.powtec.2011.08.010http://dx.doi.org/10.1016/j.powtec.2011.08.010http://dx.doi.org/10.1016/j.powtec.2011.08.010mailto:[email protected]://dx.doi.org/10.1016/j.powtec.2011.08.010http://www.sciencedirect.com/science/journal/00325910http://www.sciencedirect.com/science/journal/00325910http://dx.doi.org/10.1016/j.powtec.2011.08.010mailto:[email protected]://dx.doi.org/10.1016/j.powtec.2011.08.010

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readily reconstitute into individual nanoparticles in an aqueousenvironment, such as the lung interstitial fluid, so that they canperform their intended therapeutic functions.

The SD technique, however, necessitates the use of high tempera-tures (100 C) to achieve a fast convective drying rate required toobtain the loweff[5]. Consequently, theSD technique is not suitable forproducing nano-aggregates of thermally-sensitive drugs and theircarriers (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 success-fully produced with thehelp of dryingadjuvants,the physical integrity ofthe PCL nanoparticles was compromised due to exposures to temper-atures high above the melting point, which resulted in irreversiblenanoparticle coalescences that diminished their therapeutic functions.

In this regard, spray-freeze-drying (SFD) offers a distinct advantageover SD by eliminating the need for high-temperature operations. TheSFD technique has been widely employed in the dry-powder prepara-tion of thermally-labile proteins [7]. In the SFD process, atomizeddroplets of the nanoparticle suspension are rapidly frozen in liquidnitrogen, followed by lyophilization of the ice crystals to produce dry-powder micro-scale aggregates of the nanoparticles. Particles producedby SFD typically possess larger dg than those produced by SD, becausethe rapid freezing step, followed by sublimation in SFD, results inparticle size that is comparable to the initial droplet size, unlike in SDthat relies on droplet evaporation hence shrank particle size.

In addition, SFD particles are inherently porous yielding low effowed to the interstitial sublimation of the ice crystals in the frozendroplets. As a result, the SFD technique is highly suitable to producenano-aggregates having large dg and low eff. Our first attempt onstudying SFD of PCL nanoparticles in Cheow et al. [8] demonstratedthat large porous aggregates of PCL nanoparticles, having the desireddg (2040 m) and the desiredtheoretical da (35 m) calculatedfrom Eq. (1), were successfully produced. The effects of freeze-dryingadjuvant (only water-soluble ones) formulation on the morphology(i.e. dg, eff, and da), production yield, and aqueous reconstitution ofthe 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), wasfound necessary to produce nano-aggregates that could reconstituteinto individual nanoparticles upon exposure to aqueous media. Therole of the adjuvants here is to form interparticle bridges thatprevent nanoparticles from being in direct contact with each otherduring lyophilization. Thereby, upon dissolution of the adjuvantbridges in aqueous media, the nano-aggregates readily reconstituteinto individual nanoparticles, though the extent of reconstitution mayvary depending on the adjuvant formulation.

Despite having the desired morphology and reasonable aqueousreconstitutibility, the nano-aggregates produced in Cheow et al. [8]exhibit low fine particle fractions (FPF) (i.e. 10%), which suggestspoor particle deposition efficiency in the lung. The low FPF, which isdefined as the fraction of particles with da5 m, is caused by

agglomeration of the nano-aggregate powders upon aerosolization.The agglomeration is due to their cohesive nature attributed to themoisture uptake of the hydrophilic adjuvants. To follow up the workof Cheow et al. [8], herein we aim to improve the aerosolizationefficiency of the nano-aggregates by reducing their degree ofcohesiveness, while maintaining the particle morphology, flowability,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 ininfluencing the nano-aggregate characteristics, therefore the effects ofboth must be investigated concurrently.

In the first approach, SFD experiments employing a smaller amountof hydrophilic adjuvant than that used in Cheow et al. [8] to reduce thedegree 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 adjuvantamountwill be studied. In the second approach, besides reducing thehydrophilic adjuvant amount, inclusion of hydrophobic adjuvants isthought to be able to further reduce the degree of cohesiveness. Thepresence of hydrophobic adjuvants, however, may negatively influencethe other nano-aggregate characteristics, particularly the aqueousreconstitution. Therefore, the effects of both SFD process parametersand hydrophilichydrophobic adjuvant formulation will be concurrent-

ly examined.Low melting point PCL (62 C) is used as the thermally-sensitivepolymeric nanoparticle model. PCLhas beenwidelyinvestigated as drugcarriers due to its biodegradability, non-toxicity, and high matrixpermeability towards a wide range of drugs [9]. Levofloxacin antibioticis loaded into the PCL nanoparticles to create a model for inhalableantibiotic nanoparticles to treat respiratory infections. Mannitol andleucine,which both have acquiredgenerally-recognized-as-safe (GRAS)status, are used as the hydrophilic and hydrophobic adjuvants,respectively. Mannitol is used as the hydrophilic adjuvant, instead ofPVA, because inhaled mannitol has been found to improve mucusclearance in infected lungs [10].

2. Materials and methods

2.1. Materials

PCL (MW=80,000), levofloxacin (LEV), polyvinyl alcohol (PVA,MW= 23,000), dichloromethane (DCM) are used in the nanoparticlepreparation. D-mannitol and L-leucine are used in the SFD process.Sodium periodate, sodium thiosulfate, acetylacetone, and ammoniumacetate are used in the mannitol colorimetric assay. All chemicals usedare purchased from Sigma-Aldrich (USA).

2.2. Methods

2.2.1. PCL nanoparticle preparation and characterization

LEV-loaded PCL nanoparticles are prepared using an emulsification-solvent-evaporation method. Briefly, 8 mg of LEV and 80 mg of PCL are

dissolvedin 2 mLof DCM asthe organic phase,which isthenpoured into6 mL of 1.0% (w/v) aqueous PVA solution. The resultant mixture isemulsified for 1 min using a Vibra-Cell probe sonicator (Sonics &Materials, USA). The resultant nano-emulsion is transferred into 10 mLof 0.1% (w/v) aqueous PVA solution and stirred overnight at roomtemperature to evaporate offthe DCM resulting in theproductionof PCLnanoparticles. Afterwards, the nanoparticle suspension is centrifugedtwice at 13,000 rpmfor 12 min to remove theresidual PVAand LEV. Theoriginal nanoparticle size (Si) is measured using a Brookhaven 90PlusNanoparticle 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 nanoparticlesuspension with aqueous solution of the adjuvants. The SFD process isperformed in a modified BCHI B-290 mini spray-dryer (BCHI,Switzerland) illustrated in Fig. 1, where the drying chamber of thespray dryer is removed and replaced with a polypropylene vesselcontaining 400 mL of liquid nitrogen under constantstirring at 500 rpm.A two-fluid atomizer with 1.5 mm nozzle diameter is used because itoffers longer operating time with minimal variations than ultrasonicatomizers, which are prone to temperature rise that may affect the SFDprocess. The distance between the nozzle tip and the liquid nitrogensurface is fixed at 10 cm. After the spray-freezing step, the slurrycontaining the frozen droplets is transferred to a lyophilizationcontainer by first evaporating the excess liquid nitrogen. The frozendroplets are then lyophilized at52 C and 0.05 mbar for 24 h in Alpha

1

2 LD Plus freeze dryer (Martin Christ, Germany).

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2.2.3. Characterizations of dry-powder aggregates of PCL nanoparticles

2.2.3.1. Morphology, production yield, and flowability. The nano-aggregate morphology is characterized using scanning electronmicroscope (SEM) model JSM-6700 F (JEOL, USA). The volume-averaged dg is determined by measuring triplicates of 1000 particlesfrom the SEM images using ImageJ software (NIH, USA). The imageanalysis method is preferred over the conventional laser diffractionmethod that employs mechanical agitation and sonication prior to themeasurement to disperse the particles, which leads to the nano-aggregates being reconstituted prematurely, hence resulting in under-estimated dg values. effis determined from tap density(tap)usingatapdensitometer (Quantachromme, USA) based on 2000 taps of 1 mLpowder in two replicates. The theoretical da value is calculated fromEq. (1) using the measured values of dg and eff.

The production yield (i.e. Yield) is determined from the ratio ofnano-aggregate mass collected to the initial mass in the feedsuspension (i.e. PCL nanoparticles+adjuvants). The flowability ischaracterized by Carr's compressibility index (CI) calculated fromEq. (2), where CI values below 25 indicate good flowability and CIvalues above 40 indicate poor flowability [11]. Thebulk density (bulk)is determined by measuring the volume of powders of a known massin a 5 mL measuring cylinder without tapping. Yield and CImeasurements are performed in two replicates.

CI = 1bulk

tap ! 100% 2

2.2.3.2. Aqueous reconstitution. Briefly, 10 mg of the nano-aggregates isadded into 2 mL of deionized water under gentle stirring. Thenanoparticle size after reconstitution (Sf) is measured in triplicates.The ratio of Sf/Si indicates the extent of reconstitution, where fullyreconstituted nano-aggregates exhibit Sf/Si ratio1 [12]. To deter-mine the fraction of the nano-aggregates that have reconstituted (i.e.% reconstitution), the suspension is centrifuged at 6000 rpm for10 min after which 1.5 mL of the supernatant is discarded to remove

the reconstituted nanoparticles and the dissolved adjuvants. Thesupernatant is replaced with 1.5 mL of deionized water and thecentrifugation step is repeated three times. At the end of the fourthcentrifugation, after 1.5 mL of the supernatant is discarded, thesedimented pellet is lyophilized for 24 h. The lyophilized pellet isweighted and the % reconstitution is calculated with respect to theinitial amount of nano-aggregates supplied.

2.2.3.3. Aerosolization efficiency. The aerosolization efficiency ischaracterized using a seven-stage Next Generation PharmaceuticalImpactor (NGI; Copley Scientific, UK) equipped with an induction port(IP) and a pre-separator (PS). The aerosolization efficiency ischaracterized using two replicates. Four parameters are used toexamine the aerosolization efficiency, i.e. emitted dose (ED), fineparticle fraction (FPF), mass median aerodynamic diameter (MMAD),and cohesion index. ED is defined as the amount of powderssuccessfully aerosolized off the inhaler expressed as a percentage ofthe total amount of powders initially placed in the inhaler. FPF isdefined as the amount of powders with da5 m expressed as apercentage of the total amount of powders collected in the impactor.MMAD is defined as the median da value determined from thecumulative mass distribution plot of da. The cohesion index, whichcharacterizes the degree of nano-aggregate agglomeration uponaerosolization, is determined from the ratio of MMAD to thetheoretical da (Eq. 1), where high cohesion index indicates strongagglomeration upon aerosolization [13].

A modified powder entrainment tube (PET) based on the work ofLouey et.al. (2006) [14] is employed in place of an inhaler. Aerosoliza-tion performanceof thePET has been demonstratedto be comparable to

thatof two commercial inhalers i.e.inhalator and rotahaler [14].Theuseof PET enables evaluations of the aerosolization efficiency to beindependent of the type of inhaler used. Furthermore, the PETeliminates the dosing complexities of commercial inhalers, such as theneed to use capsules and blisters. The PET consists of three sectionsnamely inlet,powder dosage area, andoutlet (Fig. 2). A fully-developedturbulent airflow is generated in the inlet to entrain 5 mg of particlesplaced in the dosage area. A removable mesh is included in the powderdosage area to assist powder dispersions. The outlet of the PET isconnected to IP of the NGI.

Theairflow rate neededto achieve therecommended 4 kPapressuredrop over the PET is 85 L/min determined using criticalflow controller(CopleyScientific, UK). Theeffective cut-off da foreach stageat 85 L/minis6.7,3.7,2.4,1.4,0.8,0.5and0.3 m from stages 1 to 7,respectively.The

flow duration is set to 2.8 s to draw 4 L of air that simulates typicalhuman inhalation volume. The PS and impactor stages are coated withsilicone grease to prevent particle re-entrainment after deposition.Powders recovered in the IP, PS, and impactor stages are quantifiedusing a mannitol colorimetric assay [15]. From the amount of mannitol,the amount of powders recovered in each stage is determined from theknown nanoparticle to adjuvant ratio.

Briefly, 2 mL aliquot of the aqueous suspension of the recoveredpowders is centrifuged at 14,000 rpm for 12 min. The supernatant iscollected and diluted to 2 mL with deionized water. 1 mL of 5 mMsodium periodate is added to the diluted supernatant and is agitatedfor 15 s. Afterwards, 1 mL solution containing 0.1 M acetylacetone,2 M ammonium acetate, and 0.02 M sodium thiosulfate is added andagitated 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.

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wavelengths with UVVis spectrophotometer (UV Mini-1240, Shi-madzu, 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 fixed at1:1 (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/Mratios higher than 1:1 leadto poor aqueous reconstitution of the nano-aggregates therefore theyarenot pursued here. At this lowermannitolamount,the effects of threeSFD process parameters i.e. total solid concentration (TS), feedrate (FR),and atomization rate (AR) on dg, eff, da, Yield,CI,andSf/Si on the nano-aggregate characteristics are investigated by 23 factorial designs. Theresults 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 rangefrom 2.5 to 4.0% (w/v), 0.13to 0.27 L/h, and250 to 350 L/h, respectively.For comparison, Cheow et al. [8] performed their SFD experiment atfixed FR=0.24 L/h and AR= 240 L/h.

3.1.1. Effects of SFD process parameters on morphology, production yield,

and flowability

Two runs conducted at the lower limits of both FR and AR (i.e. RunsA1 and A5) produce highly fragile nano-aggregates with significantamount of fragments, hence the results are not reported here. For runsthat produce structurally robust nano-aggregates (i.e. Runs A2A4 andA6A8), the particles produced exhibit large dg and spherical shape asshown in Fig.3A. A close-uplook at the nano-aggregate surface in Fig. 3B

reveals that PCL nanoparticles, whose size (Si) is 26010 nm, arephysically dispersed in the porous mannitol matrix. The failed Runs A1and A5 prevent us from analyzing the factorial design experimentalresults to identify the statistically significant parameters. As analternative, the effects of FR and AR on dg, eff, and da are examinedseparately in Fig. 4A and B, respectively, as a function of TS. The da linesin Fig. 4 represent the theoretical da values calculated from Eq. (1) forgiven values of dg and eff. The theoretical da lines serve a purpose of

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

Table 1

A summary of PCLmannitol nano-aggregate characteristics.

Run TS(% w/v)

FR(L/h)

AR(L/h)

Yield CI Sf/Si

A1 2.5 0.13 250 Fragile aggregatesA2 2.5 0.13 350 21% 40 2.9A3 2.5 0.27 250 16% 40 2.9A4 2.5 0.27 350 29% 24 2.8A5 4.0 0.13 250 Fragile aggregatesA6 4.0 0.13 350 35% 27 2.0A7 4.0 0.27 250 30% 31 2.2A8 4.0 0.27 350 37% 26 1.3A9 5.5 0.27 410 52% 15 2.1A10 7.0 0.27 440 48% 16 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 theranges of dg and effthat yield thedesiredda range (15 m).

First, the effects of FR on dg and da are examined at a constantAR=350 L/h in Fig. 4A. At TS=2.5% (w/v) (i.e. Runs A2 and A4), theeffects of FR variation on dg and da are minimal, where particles havingdg20 m and da23 m are produced. Similar observations aremade at TS= 4.0% (w/v) (i.e.Runs A6 andA8) though the particles haveslightly larger da (34 m). The effect of FR on the production yield is

also relatively insignifi

cant, where on average the yields are equal to25% and 35% at TS=2.5 and 4.0% (w/v), respectively. The yield cantherefore be increased by increasing TS without any significant impactson dg and da.

Second, the effects of AR on dg and da are examined at a constantFR= 0.27 L/h in Fig. 4B.At TS=2.5% (w/v) (i.e. Runs A3A4), dg of theparticles are significantly reduced from50 m to 20 m when AR israised from 250 to 350 L/h, which is not unexpected as higher AR atconstant FR results in smaller droplets. The effvalues of the particles,however, remain constant at 0.02 g/cm3 despite the dg variation.

Consequently, da of particles in Run A3 (N5 m) is significantly largerthan that of Run A4 (3 m). A similar trend in the dg variation as afunction of AR is observed at TS= 4.0% (w/v) (i.e. Runs A7A8). UnlikeRuns A3A4, however, effof Run A8 is considerably higher than thatof Run A7.

Importantly, the results of Runs A3 and A7 reaffirm the earlierfinding that the productionyield can be increasedby increasing TS withminimal 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 fromsubsequent discussions. In terms of theflowability, Runs A4, A6, andA8exhibit CI values252 signifying good powder flowability. The highCIvalue inRun A2(40) is best interpreted as an experimental outlier,possibly due to sample preparation error, because the particles in RunA2 possess nearly identical dg and effas those in Runs A4 and A6, suchthat they should have exhibited similar flowability, everything elsebeing equal. Lastly, the geometric particle size distributions in Fig. 5A,represented by that of Run A8, denotes a rather wide distributionbetween 20 and 60 m, which is an inherent characteristic of SFDpowders 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 exhibitsreasonable Sf/Si1.3, whereas the other three runs (i.e. Runs A2, A4,and A6) exhibit Sf/Si2.0. The lower Sf/Si in Run A8, which possessessimilar dg, but higher effcomparedto the other three runs, suggests thedependence of aqueous reconstitution on eff. Importantly, the low Sf/Siof Run A8 obtained at P/M ratio=1:1 is comparable to that obtained inCheowetal.[8]atP/Mratio=3:7.The%reconstitutionofRunA8at605%, however, is lower than85% obtained in Cheow et al. [8].Dueto thehigherP/Mratio used here. To further investigate theeffect ofeffon Sf/Si,two additional runs (i.e. Runs A9A10) are performed at higher TS (i.e.5.5 and 7.0% w/v) aimedto increase eff, while keeping FR at 0.27 L/h. Tomaintain da15 m, AR is simultaneously increased with TS in RunsA9A10 in order to prevent dg from getting too large.

The results in Fig. 5B indicate that higher effare 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 unexpectedas TS is increased. The highereffinRuns A9A10 (0.040.045 g/cm3) also enhances the flowability (CI15)despite the smaller dg. The higher eff, however, does not lead toimprovement in Sf/Si, where the values in fact increase to 2.1 and 1.7for Runs A9 and A10, respectively, indicating poorer aqueousreconstitution. The results hence indicate that both dg and eff areimportant in obtaining the desired Sf/Si values.

3.1.3. Subsequent impacts on aerosolization efficiency

All particles produced in Runs A2, A4, A6, A8, and A9A10 exhibitsimilar values of FPF and MMAD, where they exhibit low FPF (10%)with MMAD8.0 m similar to the findings 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 thatentrainment of these particles off the PET is not an issue. Instead, thepoor FPF and MMAD are caused by the nano-aggregates formingagglomerates airborne, which are difficult to disperse, resulting in asignificant fraction of the particles (65%) recovered in the IP and PSof the NGI (Fig. 6).

In summary, SFD at a lower mannitol content (i.e. higher P/Mratio) can produce highly reconstitutable nano-aggregates having thedesired dg and theoretical da values, as well as good flowability andproduction yield, provided that the SFD process parameters, partic-ularly TS and AR, are optimized, as exemplified by Run A8. However,the lower mannitol content,doesnot lead to any improvements in theaerosolization efficiency. As the P/M ratio cannot be increased further,the effects of multiple excipient formulations involving both hydro-

philic 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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3.2. Second approach by inclusion of both hydrophilic and hydrophobic

adjuvants

Owing to the hydrophobic nature of leucine, its inclusion isanticipated to limit the moisture uptake of the PCLMannitol nano-

aggregates, which would render the particles less cohesive henceimproving the aerosolization efficiency. Furthermore, the presence ofleucine on particle surfaces has also been found to lower the surfacefree energy therefore reducing the particle tendency to agglomerate[16]. In addition, leucine inclusion is known to produce particles withcorrugated surfaces upon drying, which reduce interparticle contactsurface areas, resulting in better particle dispersions [13].

3.2.1. Effects on morphology, production yield, flowability, and aqueousreconstitution

The effects of incorporating leucine into the PCLMannitolformulation on dg, eff, da, Yield, CI, and Sf/Si are investigated inRuns B1B6 presented in Table 2. The investigations are performed atconstant FR=0.27 L/h as the effects of FR have been found earlier tobe insignificant. In Runs B1B6, the effects of varying the mannitol toleucine concentration ratio (i.e. M/L ratio) at different TS areinvestigated for M/L ratios of 6:1 and 5:2. The PCL nanoparticleconcentration is fixed at one-eighth of TS because higher nanoparticleconcentrations would require even higher M/L ratios to reconstitutethe nano-aggregates.

The high M/L ratio is required because spray-freeze-dried leucineparticles are highly fragile [8], such that when leucine is included inthe formulation to improve the aerosolization efficiency, a largeramount of mannitol is needed to maintain the structural integrity ofthe nano-aggregates. In addition, the presence of leucine decreasesthe aqueous reconstitution due to the low aqueous solubility ofleucine (25 mg/mL) compared to 180 mg/mL for mannitol.Consequently, a larger amount of mannitol is needed for reconstitu-tion to counteract the presence of leucine.

Starting at TS=4.0% (w/v), which is found earlier to be optimal inthe PCLMannitol formulation, spherical particles (Fig. 7A), withdg14 m and eff0.02 g/cm

3 resulting in da2 m, are successfullyproduced at AR= 410 L/h (i.e. Runs B1B2). Because of the largerpresence of mannitol, which tends to produce large particles, higher ARis needed to maintain dg within the 1030 m range, so that particleswith theoretical da15 m can be obtained. A close-up view of thePCLmannitolleucine nano-aggregates in Fig. 7B denotes a highly

corrugated surface attributedto theleucineinclusion.Not unexpectedly,the CI values (34) indicate reasonably good flowability owed to thecorrugated surface, despite the high mannitol content. The productionyield, however, is rather low at20%. The effects of M/L ratio in RunsB1B2ondg, eff,andda (Fig. 8), aswell ason the Yield and CI, are foundto be minimal. The higher M/L ratio in Run B1 nonetheless results inlower Sf/Si comparedto RunB2 (i.e. 1.2 versus 1.9) denoting reasonablereconstitutibility.

To increase the production yield, TS is increased to 5.5% (w/v) atAR=440 L/h in Runs B3B4. The production yield is increased to4050% as expected. The effects of increasing TS from 4.0 to 5.5%(w/v) on dg, da, and CI are fairly insignificant, 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/Si1.3 denoting good reconstitutibilitywith % reconstitutions above 60%. In contrast, the effect of M/Lratio ondg is intensified, where the lower M/L ratio produces considerably

0

10

20

30

0 20 40 60 80

%v

olume

A8

A

B

0.05

0.04

0.03

0.02

0.01

0

eff(g/cm

3)

5 15 25 35 45 55dg (m)

dg (m)

2 m

da = 1.5 m

3 m4 m 5 m

A9

A10

A8

FR = 0.27 L/h

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

Fig. 6. Typical particle deposition patterns of PCL

mannitol nano-aggregates.

Table 2

A summary of PCLmannitolleucine nano-aggregate characteristics.

Run TS(% w/v)

PCL(% w/v)

AR(L/h)

M/L Yield CI Sf/Si

B1 4.0 0.5% 410 6:1 20% 34 1.2B2 5:2 21% 34 1.9B3 5.5 0.7% 440 6:1 37% 31 1.3B4 5:2 49% 33 1.3B5 2.4 0.3% 350 6:1 36% 30 1.8B6 5:2 35% 22 1.8

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smaller dg. The effects of M/L ratio on the rest of the parametersremain relatively insignificant. In terms of the particle size distribu-tion, the nano-aggregates produced in Runs B3B4 exhibit similar sizedistributions as that presented in Fig.5A,hencetheyare not presentedhere for brevity.

For comparison, at a lower TS = 2.4% (w/v) and AR= 350 L/h inRuns B5B6, slightly larger particles having lower eff are produced

(Fig. 8). The nano-aggregates are poorly reconstituted as manifestedinthe highS f/Si values (1.8), regardless of the M/L ratios. The effectsof M/L ratio on dg, eff, Yield, and CI are also insignificant. Theproduction yield nonetheless is unexpectedly higher than thatobtained at TS= 4.0% (w/v). Taking into account the particlemorphology, production yield, flowability, and aqueous reconstitu-tion, Runs B3B4 are determined as the optimal SFD run for the PCLMannitolLeucine formulation.

3.2.2. Effects on aerosolization efficiency

The ED, FPF, MMAD, and cohesion index values of the particlesproduced in Runs B1B6 are summarized in Table 3. The experimentaluncertainties in ED, FPF, MMAD, and cohesion index based on tworeplicates are 8%, 3%, 3%, and 11%, respectively. All six runsdemonstrateED90%that signifies highly effective particle entrainmentoff the PET, which was also observed with the PCLmannitol nano-aggregates. Significantly, the leucine inclusion enhances the FPF andMMAD as postulated. RunswithM/Lratio= 5:2(i.e.Runs B2,B4, andB6)exhibit higher FPF and lower MMAD values compared to those of Runswith M/L ratio=6:1 (i.e. Runs B3, B5, and B7). Therefore, the higher theleucine content, the higher (lower) the FPF (MMAD).

Therepresentativeparticledeposition patterns of Runs B2,B4, and B6are provided in Fig. 9. A significant fraction of particles are recovered instages 24 representing vast improvement over the PCLmannitolformulation presented in Fig. 6. The cohesion index, however, remainshigh ranging from2.0 to 3.7denoting still significant agglomeration ofthe nano-aggregates,despite the leucine presence. Notably,runswiththesame M/Lratio exhibitsimilar FPF and MMAD valuesirrespective of their

dg and eff values, which signify that the particle morphology has lessimpact towards aerosolization efficiency than the adjuvant formulation.

Taking all factors into consideration, Run B4 clearly represents theoptimal SFD process parameters and adjuvant formulation, whereYield50%, Sf/Si1.3, ED96%, and FPF30% are obtained. The highFPF and low MMAD, which are comparable to the values exhibited bycommercial DPI, are undeniably obtained at the expense of havinghigher adjuvant to nanoparticle ratios, particularly of mannitol.

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

da= 1.5 m2 m 3 m 4 m

B5B6

B1

B2

B3

B4

0

0.01

0.02

0.03

0.04

5 15 25 35

eff(g/cm

3)

dg (m)

Fig. 8. Effects of different PCL

mannitol

leucine formulations on dg, eff, and da.

Table 3

Aerosolization characteristics of PCLmannitolleucine nano-aggregates.

Run B1 B2 B3 B4 B5 B6

ED 88% 92% 92% 96% 94% 95%FPF (5 m) 22% 32% 20% 30% 17% 29%MMAD 6.5 5.3 7.2 5.5 7.8 5.3Cohesion index 3.4 2.9 2.5 3.4 3.7 2.0

Fig. 9. Typical particle deposition patterns of PCLmannitolleucine nano-aggregates in

Runs B2, B4, and B6.

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However, it has to be pointed out that, unlike the conventional DPIdelivery, these nano-aggregates are readily aerosolized without theassistance of coarse carrier particles (e.g. lactose) owed to their uniquemorphology (i.e. large dg and low eff). As a result, the amounts ofadjuvants used exclusively for delivery purposes have been essentiallyreduced compared to the conventional delivery mode. From thatperspective, the carrier-free aerosolization here can adequately justifyfor the high adjuvant to nanoparticle ratio required.

4. Conclusion

Two studies aimed to improve the aerosolization efficiency ofspray-freeze-dried aggregates of thermally-sensitive polymeric nano-particles have been performed via optimizations of both the SFDprocess parameters and adjuvant formulations. In addition to theaerosolization efficiency, the optimization also takes into account theparticle morphology, production yield, flowability, and aqueousreconstitution. In the first study, the hydrophilic adjuvant content(i.e. mannitol) is lowered to the minimal amount below which thenano-aggregates cannot sufficiently reconstitute aimed to reduceparticle cohesiveness. Poor aerosolization efficiency as manifested bythe low fine particle fractions (10%), however, remains observedindependent of the other nano-aggregate characteristics. In thesecond study, the inclusion of both hydrophilic (i.e. mannitol) andhydrophobic (i.e. leucine) adjuvants significantly improves the fineparticle fraction (30%), without jeopardizing the other nano-aggregate characteristics, as long as the SFD process parameters andmannitol to leucine ratio are optimized. The particle size distribution,however, still leaves much room for improvement in terms of themonodispersity. Experimental works on improving the particle sizedistribution by controlling the sprayed droplet size are currentlyongoing in our laboratory.

Nomenclature

AR atomization flow rate (L/h)da aerodynamic diameter (m)dg geometric diameter (m)

ED emitted dose (% w/w)FPF fine 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 indexSi raw nanoparticle size (nm)Sf nanoparticle size after aqueous reconstitution (nm)

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

Acknowledgment

The authors would like to thank Jellynn Teng and Prima Dewi

Sinawang for their contributions in the aerosolization efficiencycharacterization study. Financial supports from Nanyang TechnologicalUniversity's Start-Up Grant (SUG 8/07) and Undergraduate ResearchExperience on Campus (URECA) are gratefully acknowledged.

References

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