Powder Technology 237 (2013) 468–476

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Particle size reduction of poorly water soluble artemisinin via antisolventprecipitation with a syringe pump

Mitali Kakran a, Nanda Gopal Sahoo a, Lin Li a,⁎, Zaher Judeh b

a School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singaporeb School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore

⁎ Corresponding author. Tel.: +65 6790 6285; fax: +E-mail address: mlli@ntu.edu.sg (L. Li).

0032-5910/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.powtec.2012.12.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 August 2012Received in revised form 6 December 2012Accepted 15 December 2012Available online 22 December 2012

Keywords:ArtemisininAntisolvent precipitationSyringe pumpCrystallinityParticle sizeDissolution

The method called antisolvent precipitation with a syringe pump (APSP) was used for reducing the particle sizeof a poorly water soluble anti-malarial drug, artemisinin (ART) with the aim of improving its dissolution proper-ties. Various process parameters, such as drug concentration, solvent–antisolvent volume ratio, stirring speed,flow rate and temperature were investigated and optimized to produce the smallest particle size. As part ofthe design of experiment, a percent dissolution surface response model was regressed and statistically assessedto understand the relationship between the process parameters and percent dissolution. The particle size of thecommercial ART was reduced from 26.4 μm (diameter) and 30.0 μm (length) to 1.5 μm (diameter) and 3.8 μm(length) by the APSPmethod, which increased the percent dissolution of ART. The DSC and XRD studies revealedthat the crystallinity of ART particles preparedwas lower than the commercial ART. The XRD study also revealedthe fabrication of two polymorphs of ART, i.e. the orthorhombic and triclinic form. Commercial ART and ART par-ticles fabricated by APSP (in the absence of polymers)were orthorhombicwhereas ART prepared in the presenceof a polymer, polyvinylpyrrolidone or polyethylene glycol, was of triclinic form.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Artemisinin is a promising antimalarial drug that remains effectiveagainst multidrug resistant and cerebral malaria-causing strains ofPlasmodium falciparum. It can also aid in treating other infectious dis-eases such as schistosomiasis, leishmaniasis, hepatitis B and numeroustypes of tumors and cancer cell lines [1,2]. It has good intestinal perme-ability and readily crosses the intestinal monolayers via passive diffu-sion [3]. Despite these medicinal benefits, the major problem withartemisinin (ART) is its poor aqueous solubility [4], resulting in poor ab-sorption upon oral administration. This poor solubility, its short half lifeand high first-pass metabolism, might lead to incomplete clearance ofthe parasites resulting in recrudescence [5]. All these highlight theneed for an improved formulation for ART with enhanced dissolutionso that its absorption can be greatly enhanced. The solution is to de-crease the particle size of ART. According to the Noyes–Whitney equa-tion [6], a decrease in particle size will lead to an increase in effectivesurface area, which, in turn, increases the drug dissolution rate. The im-proved dissolution rate of the drug in-vitrowill result in enhanced bio-availability of the drug in-vivo.

There are many ways to achieve smaller particles: for example me-chanical processes, precipitation methods, etc. The mechanical process,e.g., milling and homogenization, use shear or particle collisions as the

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energy source to break down larger entities into smaller ones. The pre-cipitation process, such as antisolvent precipitation, is quite simple, costeffective and easy for scaling-up to produce nanoparticles of poorlywater soluble drugs by mixing a drug solution and an antisolvent[7,8]. Therefore, in this study, antisolvent precipitation process wasmodified and developed as themethod called the antisolvent precipita-tion with a syringe pump (APSP) to prepare smaller drug particles ofART to improve its dissolution rate.

Precipitation consists of generation of supersaturation, nucleation,and subsequent growth of nuclei. The driving force for precipitation,called supersaturation (S), is defined as:

S ¼ Co

C� ð1Þ

where Co is the initial concentration of a drug in the solution to beprecipitated and C* is the solubility of the drug in the solvent–antisolvent system. The Gibbs free energy change, ΔG, is the energybarrier that a nucleation process must overcome [9]:

ΔG ¼ 16πγ3V3S

3k2T2 lnSð Þ2 ð2Þ

whereγ is the surface tension,VS is themolecular volume (molar volume/Avogadro number), k is the Boltzmann constant, T is the absolute temper-ature, and S is the supersaturation. According to Eq. (2), a higher supersat-uration (S) and a lower surface tension (γ) result in a lower critical energy

469M. Kakran et al. / Powder Technology 237 (2013) 468–476

barrier (ΔG), which leads to fast nucleation and hence, production ofsmaller particles [8,10]. The kinetic parameter describing how fast the nu-cleation occurs is given by the rate of nucleation per unit volume and perunit time, RN, as [9]:

RN ¼ CokT3πλ3η

� �exp −ΔG

kT

� �ð3Þ

where λ is the diameter of the growth species, and η is the viscosity of thesolution. This equation indicates that high initial concentration (Co), lowviscosity (η) and low critical energy barrier (ΔG) favor the formation ofa large number of nuclei. At a given concentration of a solute, a largernumber of nuclei mean smaller nuclei.

Various process parameters, such as drug concentration, solvent toantisolvent (SAS) volume ratio, stirring speed, and flow rate havebeen investigated and optimized to produce the smallest drug particlesize. The properties of the ART particles fabricated have been studiedin terms of particle size and morphology, crystallinity and the dissolu-tion rate. The effect of temperature as well as the influence of addinga water-soluble polymer such as polyvinylpyrrolidone (PVP) or poly-ethylene glycol (PEG) upon the precipitation of ART particles duringthe APSP process has also been investigated. The interesting findingsabout the changes in the morphology and crystal structure of ARTformed in the presence of a polymer (PVP or PEG) during the APSPmethod are reported. The polymorphic form of a drug can have a con-siderable effect on its pharmaceutical effectiveness, particularly whenthe dissolution rate of a drug determines its absorption in the gastroin-testinal tract. Previously Chan et al. reported the X-ray crystallographicevidence for the existence of the two ART polymorphs—triclinic and or-thorhombic polymorphs [11]. Both forms of the polymorphs of ARThave been fabricated and studied in this work.

2. Materials and methods

2.1. Materials

Artemisinin (ART) was obtained from Kunming PharmaceuticalCorporation (Kunming, China). Polyvinylpyrrolidone (PVP) and poly-ethylene glycol (PEG) were obtained from Sigma Aldrich (Singapore).All the reagents used were of technical grade.

2.2. Methods

Commercial ART was dissolved in the solvent (ethanol) at concen-trations ranging from 5 to 15 mg/ml. The syringe was filled with theprepared solution and secured onto a syringe pump. Drug solutionwas quickly injected at a flow rate (from 2 to 10 ml/min) into theantisolvent (deionized water) under magnetic stirring (200 to1000 rpm). The solvent to antisolvent volume ratios used were 1:10,1:15 and 1:20. The ART particles precipitated were filtered and vacuumdried. To investigate the effect of a water-soluble polymer (PVP andPEG), PVP or PEG was dissolved in water at the concentration of5 mg/ml and then used as the “antisolvent”. The rest of the procedurewas the same as above to obtain ART particles with PVP or PEG.

2.3. Particle morphology

The particle size and morphology of samples were observed usinga scanning electron microscope (JSM-6390LA-SEM, Jeol Co., Japan).The powder samples were spread on a SEM stub and sputtered withgold before the SEM observations. The analysis of the particle sizewas performed using the UTHSCSA ImageTool program.

2.4. DSC analysis

Differential scanning calorimetric (DSC) measurements were car-ried out using a TA DSC 200 thermal analyzer in a temperaturerange of 50–200 °C at a heating rate of 10 °C/min in nitrogen gas.The melting points were determined and the heats of fusion were cal-culated using the DSC software.

2.5. X-ray diffraction analysis

X-ray diffraction was analyzed using the Bruker AXS D8 AdvanceX-ray diffractometer with Cu Kα—targets at a scanning rate of 0.0102θ/s, applying 40 kV, 40 mA, to observe the crystallinity of samples.

2.6. Dissolution studies

The in vitro dissolution of the ART samples prepared as well as thecommercial ART was determined using the paddle method (USP ap-paratus II) (Verkin Dissolution Tester DIS 8000) in 100 mL of distilledwater. The paddle rotation was set at 100 rpm. The temperature wasmaintained at 37±0.5 °C. APSP prepared samples and original ARTpowder containing 30 mg of ART were used for the dissolution exper-iments. Samples of 1 ml volume were collected at 0.5, 1, 2, 3 and4 hour intervals. The tests were repeated 3 times and the dissolutiondata was averaged.

2.7. Analysis of ART concentration

The ART concentration for the dissolution studies was determinedusing a high performance liquid chromatography (HPLC) method withultraviolet (UV) detection [12]. The HPLC used was Agilent 1100 seriesequipped with the column of Kromasil C18 (150 mm×4.6 mmid×3.5 μm) (Eka Chemicals AB, Sweden). The mobile phase consistedof 75% of 0.01 M disodium hydrogen phosphate and 25% acetonitrile(HPLC grade), and it was adjusted to pH 6.5 with glacial acetic acid.The flow rate was set at 0.8 mL/min. The UV detector was operated ata wavelength of 254 nm. The samples were filtered through 0.45 μmpolypropylene-reinforced PTFEmembranewith polypropylene housing(Ministart-SRP 15, Saritorius, Germany). The sampleswere subjected topretreatment prior to injection into the HPLC system. 1 mL of samplewas added into 200 μL of 10 M sodium hydroxide and the mixturewas heated at 45 °C for 25 min,whichwas then cooled to room temper-ature. Finally, 150 μL of glacial acetic acid was added into the abovemixture before injection into the HPLC system.

2.8. Experimental design and surface response modeling

The regression and statistical analyses for the experimental designand surface response modelling were carried out using MODDE(Umetrics, Umeå, Sweden). The Optimizer function within MODDEwas used to optimize the experimental variables for maximum disso-lution criteria. Four APSP process variables, viz. drug concentration,solvent to antisolvent ratio, stirring speed and flow rate, were consid-ered. A full factorial Central Composite Face centered (CCF) experi-mental design was used to generate 25 runs (Table 1). The rangeand level of factors used are shown in Table 2. A Multilinear Regres-sion Model was fitted to the four independent variables (X) and onedependent response (Y), which is dissolution after 4 h.

2.9. Statistical analysis

Relative dissolution (RD) and percent dissolution efficiency (%DE)were calculated to compare the relative dissolution performance ofthe various samples [13]. RD was the number of times the dissolutionincreased with respect to the reference sample (original drug). The%DE for each formulation was computed as the percent ratio of area

Table 1Various ART samples prepared by the APSP method along with the range of four independent variables: drug concentration (mg/ml), solvent to antisolvent volume ratio (SAS),stirring speed (rpm), flow rate (ml/min), their particle size, and their observed and predicted percent dissolution (%) at 4 h.

Exp. no. Drug conc. SAS ratio Stirring speed Flow rate C⁎ (μg/ml) S Re Particle size (μm) Dissolution (observed) Dissolution(predicted)

Diameter Length

Original ART 26.4 30.00 13.10±2.11 –

1 5 1:10 200 2 40.1 124.7 144 8.6 21.0 35.19±3.32 33.61±1.992 15 1:10 200 2 40.1 374.1 144 7.2 22.5 30.20±4.53 29.80±2.023 5 1:20 200 2 16.4 304.9 144 5.2 20.8 36.11±2.88 35.18±1.034 15 1:20 200 2 16.4 914.6 144 5.3 21.7 33.08±3.54 32.22±1.445 5 1:10 1000 2 40.1 124.7 144 5.6 12.2 42.21±2.21 42.94±2.616 15 1:10 1000 2 40.1 374.1 144 4.8 12.1 42.72±4.36 42.03±1.647 5 1:20 1000 2 16.4 304.9 144 2.8 7.0 50.22±3.22 50.05±2.218 15 1:20 1000 2 16.4 914.6 144 2.4 6.2 51.04±3.87 50.00±1.929 5 1:10 200 10 40.1 124.7 720 6.2 17.3 32.09±1.90 33.25±1.4510 15 1:10 200 10 40.1 374.1 720 7.1 18.3 30.21±2.42 30.00±1.2111 5 1:20 200 10 16.4 304.9 720 5.8 16.2 34.88±2.77 35.22±2.1512 15 1:20 200 10 16.4 914.6 720 5.9 17.1 33.38±1.97 32.82±2.3113 5 1:10 1000 10 40.1 124.7 720 3.4 10.2 45.72±2.67 46.23±1.2714 15 1:10 1000 10 40.1 374.1 720 3.8 11.4 44.81±5.03 45.88±1.4515 5 1:20 1000 10 16.4 304.9 720 2.2 5.8 53.06±4.56 53.75±2.1316 15 1:20 1000 10 16.4 914.6 720 2.2 5.4 53.25±5.10 54.24±1.8517 5 1:15 600 6 34.2 146.2 432 4.0 12.6 41.81±3.04 41.17±1.5718 15 1:15 600 6 34.2 438.6 432 4.1 15.1 38.15±3.91 39.51±2.6519 10 1:10 600 6 40.1 249.4 432 4.2 16.2 34.60±3.43 33.96±2.1320 10 1:20 600 6 16.4 609.8 432 4.1 15.7 37.55±4.65 38.92±1.6421 10 1:15 200 6 34.2 292.4 432 7.5 19.5 27.82±3.11 30.90±1.6822 10 1:15 1000 6 34.2 292.4 432 3.1 7.8 48.61±2.92 46.28 ±1.9623 10 1:15 600 2 34.2 292.4 144 7.8 16.5 30.18±2.78 34.97±1.4724 10 1:15 600 10 34.2 292.4 720 3.9 12.8 41.04±3.62 36.91±2.9725 10 1:15 600 6 34.2 292.4 432 4.0 14.1 39.32±2.09 36.95±3.04Low Tempa 15 1:20 1000 10 4.0 3750 720 1.5 3.8 58.21±4.11 –

PVPb 15 1:20 1000 10 16.4 914.6 720 Flake/plate type 64.13±3.19 –

PEGb 15 1:20 1000 10 16.4 914.6 720 Flake/plate type 62.02±4.82 –

a Low Temp—Experiment conducted at temperature (10 °C) lower than the room temperature.b Experiment conducted in the presence of polymers (PVP and PEG) in the antisolvent (water).

470 M. Kakran et al. / Powder Technology 237 (2013) 468–476

under the dissolution curve up to the time t, to that of the area of therectangle described by 100% dissolution at the same time:

%DE ¼∫t

0

y:dt

y100⋅t

0BBBB@

1CCCCA100 ð4Þ

The difference factor (f1), evaluating the percent error betweentwo curves over all time points, was calculated by [14]:

f 1 ¼ ∑ni¼1 Ri−Tij j∑n

i¼1Ri

� 100 ð5Þ

where n is the dissolution sample number, and Ri and Ti are the amountsdissolved of the reference and test sample at each time point i respec-tively. The percent error is zero when the test sample's and drugreference's profiles are identical and increases proportionally with thedissimilarity between the two dissolution profiles. The similarity factor(f2) is a logarithmic transformation of the sum-squared error of

Table 2Experimental design schedule showing the variables along with their range and designlevel values.

Variable Unit Range Design levels

Drug concentration (X1) mg/ml 5–15 5 10 15Solvent to antisolvent ratio (X2) – 1:10–1:20 1:10 1:15 1:20Stirring speed (X3) rpm 200–1000 200 600 1000Flow rate (X4) ml/min 2–10 2 6 10

differences between the test Ti and reference Ri over all time points,and is given by [14]:

f 2 ¼ 50� log 1þ 1=nð Þ∑ni¼1 Ri−Tij j2

h i−0:5 � 100� �

ð6Þ

It is 100 when the test and reference profiles are identical andtends to 0 as the dissimilarity increases.

2.10. Dissolution rate constant studies

The Noyes–Whitney equation provides a general guideline as tohow the dissolution rate of an insoluble drug might improve [6].The dissolution rate equation based on mass is expressed as follows:

dmdt

¼ K MS−mð Þ ð7Þ

wherem is the dissolution amount of drug at time t, dm/dt is the disso-lution rate,MS is the dissolution amount at infinite time, and t is the dis-solution time. Integrating Eq. (7) with the initial condition of m=0 fort=0, then Eq. (8) is obtained.

m ¼ MS 1− exp −Ktð Þ½ � ð8Þ

Or

mMS

¼ 1− exp −Ktð Þ½ � ð9Þ

where the dissolution rate constant K is defined as AD/h, where A is thesurface area available for dissolution, D is the diffusion coefficient of thedrug, and h is the thickness of the diffusion boundary layer adjacent tothe surface of the dissolving drug.

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3. Results and discussion

3.1. SEM analysis

The SEM photographs are shown in Fig. 1 and the description of allthe samples prepared is provided in Table 1. The original ART particles(Fig. 1(a)) were not uniform and were large in size (26–30 μm). TheART particles prepared by the APSP method, exhibited the rod-typemorphology with uniformity in size and absence of large particles. Theparticle size and morphology of the various samples prepared undervarying process parameters are discussed in the following sectionsalong with the mechanism behind.

As discussed earlier, the supersaturation is the prerequisite for nu-cleation; therefore, the values of supersaturation (S) were calculatedusing Eq. (1) and are listed in Table 1. As seen from Table 1, the valuesof S were affected by the drug concentration in the solution, the sol-vent to the antisolvent ratio (SAS) and the temperature. From the cal-culations, a larger SAS ratio, a higher drug concentration and a lower

Fig. 1. SEM photographs of ART particles prepared by APS

temperature resulted in a greater S, which principally should lead toan increased nucleation rate and hence, more nuclei with smallersizes. However, it can be seen from Table 1 that S is not the only pa-rameter that governs the kinetics of nucleation. Besides the supersat-uration that determines the nucleation, the crystal growth rate is alsoanother important factor that affects the particle size. Therefore, inthis study various critical process parameters such as stirring speed,flow rate, solvent to antisolvent ratio, drug concentration in the solvent,and temperature of the antisolvent have been examined and discussedbelow.

3.1.1. Effect of the drug concentrationThe drug concentration affects the size of precipitated particles in an

opposing way: a higher drug concentration leads to a greater supersat-uration (S) as seen fromTable 1, which results in a faster nucleation rateand thereby smaller particles. However, a higher S also speeds up ag-glomeration through the greater chance of particle collision to producebigger particles [10]. As seen from Table 1, at a lower stirring speed

P. The experiment conditions are described in Table 1.

472 M. Kakran et al. / Powder Technology 237 (2013) 468–476

(200 rpm), the size of ART particles increased slightly as the concentra-tion increased from 5 to 15 mg/ml, for example, samples 3 & 4, andsamples 11 & 12. This indicates that agglomeration prevailed over nu-cleation at high drug concentrations. This result was governed by twofactors: the number of nuclei formed at the solvent/antisolvent inter-face, and the influence of drug concentration on the viscosity [15]. Onthe one hand, at a higher concentration, a larger number of nucleiwere formed at the interface of two phases which led to agglomerationand thus, formation of larger particles. Simultaneously, those nucleiobstructed the further diffusion of drug molecules from the solvent toantisolvent. On the other hand, the viscosity of the drug solution in-creased with increasing concentration, which hindered the drug diffu-sion between solution and antisolvent, resulting in non-uniformsupersaturation [16] and thus formation of the larger drug particles.However, this trend was reversed at the higher stirring speed(1000 rpm),where the size of ART particles decreased as the concentra-tion increased from 5 to 15 mg/ml, e.g., samples 7 & 8 (Fig. 1), samples15 & 16. This suggests that as mixing is improved at a higher stirringspeed, the greater supersaturation effect dominates the agglomerationeffect of drug concentration as discussed above. Hence, at the higherstirring speed the smaller particles were produced at even higherdrug concentrations.

3.1.2. Effect of the solvent to antisolvent (SAS) volume ratioThe average particle sizes in Table 1 and the corresponding SEM

images in Fig. 1 suggest an inversely proportional relationship be-tween the amount of antisolvent in the SAS ratio and the particlesize. It can be examined from Table 1 that as the SAS volume ratiowas increased from 1:10 to 1:20, the particle size was drastically de-creased. As can be noted from Table 1, the particle size was reduced asthe SAS was increased from 1:10 to 1:20, e.g., from samples 1 to 3,samples 2 to 4, samples 5 to 7, samples 6 to 8 (shown in Fig. 1), etc.There are a few reasons for this observation. When the ART solutionwas added to the deionized water, the ART concentration was re-duced more quickly as the amount of antisolvent was increased,which led to faster precipitation of the drug into nanoparticles.From Table 1, the S values at the greater antisolvent amount werehigher, and according to Eqs. (2) & (3) a larger amount of antisolventshould lead to a greater nucleation rate and hence smaller nuclei [17].Once the nuclei are formed, growth occurs simultaneously. For thesubsequent growth, more antisolvent amount increases the diffusiondistance for growing species and consequently diffusion becomes thelimiting step for nuclei growth [18]. Therefore, the particle size de-creases as the SAS ratio increases.

3.1.3. Effect of the stirring speedStirring speed was another important factor that affected the par-

ticle size dramatically. It was observed from Table 1 that the size ofART particles decreased significantly with increasing stirring speedfrom 200 to 1000 rpm, e.g., samples 1 & 5, samples 2 & 6 (shown inFig. 1), samples 3 & 7, samples 4 & 8, samples 10 & 14 (Fig. 1), etc.The decrease in particle size of the ART particles with increase in stir-ring speed is due to the intensification of the micromixing (i.e. mixingon the molecular level) between the multi-phases [16]. Highmicromixing efficiency increased the mass transfer and the rate ofdiffusion between the multiphases, which induced high homogenoussupersaturation in short time and thus rapid nucleation to producesmaller drug particles. In addition to that, a high stirring speed alsoprevents the growth of particle by preventing their aggregation.Hence, the formation of smaller and more uniform drug particles isfavored with a higher stirring speed.

3.1.4. Effect of the flow rateAt a lower flow rate, the amount of solvent/antisolvent mixing be-

came less per unit time. Hence there were few nucleating sites, whichprolonged crystal growth processes, thus resulting in the formation of

larger crystals. As the flow rate was increased, the ART solution wasmixed more rapidly into deionized water. Increasing the flow rate in-creased the jet velocity and, hence, the Reynolds number (Re,Table 1), resulting in an increased extent of mixing between the drugsolution and the antisolvent per unit time. Since the time was shortfor allowing the crystal growth, only smaller crystals were formed[19]. The corresponding Reynolds number Re was calculated accordingto the following equation:

Re ¼VDv

¼ QDAv

ð10Þ

whereV is the flowvelocity for the solvent (m/s),D is the diameter of thepipe (m), v is the kinematic viscosity (m2/s),Q is the volumetricflow rate(m3/s) and A is the pipe cross-sectional area (m2). The diameter of theinjection needle used was 0.24 mm and the kinematic viscosity of etha-nol, 1.23×10−6 m2/s, was used to calculate the value of Re. The volu-metric flow rate, Q, was varied from 2 ml/min (3.33×10−8 m3/s) to10 ml/min (16.66×10−8 m3/s) and the corresponding values of Re in-creased from 144 to 720 (shown in Table 1). As the flow rate was in-creased from 2 to 10 ml/min, the particle size decreased from samples1 to 9, samples 2 to 10 (shown in Fig. 1), samples 7 to 15, samples 8 to16 (Fig. 1), etc.

3.1.5. Effect of temperatureTo study the effect of temperature on the size of ART particles, the

cold DI water was used as antisolvent, which was kept in an ice bathto maintain the temperature at 10 °C. The precipitation process isexpected to be influenced by the temperature through several ways.Firstly, reduction in the antisolvent temperature decreases the equilib-rium solubility of ART in the solute–solvent–antisolvent mixture andhence, increases the supersaturation. As shown in Table 1, a change inthe antisolvent temperature from room temperature (25 °C) to 10 °Cenhanced the value of S from 914.6 to 3750, so that the nucleationrate increased and hence the particle size decreased. Secondly, the pre-cipitation in the liquid phase is a diffusion-limited process [20]. At alower temperature the diffusion rate is decreased and accordingly thecrystal growth rate is slower. The decrease in the antisolvent tempera-ture from 25 to 10 °C increased the viscosity of water from 0.894 cP to1.308 cP, which led to a reduction in particle collision frequency andresulted in a decrease in particle growth by preventing coagulationand agglomeration. Therefore, the smaller particleswere formed as a re-sult of the high nucleation rate and low growth rate at the low temper-ature, 10 °C. It should be noted that, although a lower viscosity isdesired for a higher rate of nucleation to facilitate diffusion of drugfrom solvent to antisolvent, a higher viscosity would lead to lesser crys-tal growth. The particle diameter and length of the sample fabricated atthe low temperature were 1.5 μm and 3.8 μm, respectively. These arethe smallest ART particles fabricated by the APSP method as can beseen from Fig. 1(j). From these results, it can be concluded that theART particles fabricated by the APSP method were significantly smallerand more uniform than the original ART. The APSP conditions thatresulted in the smallest ART particles were: drug concentration of15 mg/ml, SAS volume ratio of 1:20, stirring speed of 1000 rpm, flowrate of 10 ml/min and temperature of 10 °C.

3.1.6. Effect of the polymerA water-soluble polymer, such as polyvinylpyrrolidone (PVP) and

polyethylene glycol (PEG), was dissolved in DI water (the antisolvent)and its effect on ART precipitation was investigated. A very interestingobservation was made regarding the morphology of the ART particles.As seen from Fig. 1(k) and (l), the ART particles were more flake orplate type instead of the rod type as obtained in the previous cases(Fig. 1(b)–(g)). The FTIR study (not shown) confirmed the absence ofthe polymers, PVP and PEG, in the ART samples and revealed no interac-tion between ART and the polymers. It is considered that the flake or

473M. Kakran et al. / Powder Technology 237 (2013) 468–476

plate type ART particles is a different polymorph of ART. PreviouslyChan et al. reported the X-ray crystallographic evidence for the exis-tence of the two polymorphs of ART: triclinic and orthorhombic poly-morphs [11]. The orthorhombic crystal had a denser and thickerappearance like rods and prisms, whereas the triclinic form mainlycontained thin blades and plates. From Fig. 1, it can be predicted thatthe original ART powder and the ART particles prepared in the absenceof a polymer were the orthorhombic form of ART, whereas the ART par-ticles precipitated in the presence of a polymer, PVP or PEG, were thetriclinic form. This can be confirmed by the XRD study.

3.2. X-ray diffraction analysis

The X-ray diffraction patterns of the original ART and the ART parti-cles fabricated by APSP with and without a polymer (PVP or PEG) werestudied in order to understand the crystallinity of the samples and thenature of the polymorphs of ART as well. The x-ray diffraction patternsof all the samples are shown in Fig. 2. The ART particles prepared by theAPSP method exhibited the lower peak intensity, indicating a certainloss in the crystallinity of the ART particles. To conclude, it can be saidthat the crystalline nature of ART was still maintained, but the relativereduction of diffraction intensity of ART in the prepared samples sug-gests that the crystallinity was reduced. The orthorhombic form ofART in Fig. 2 displayed the numerous distinct peaks at 2θ of 7.29°,11.78°, 14.65°, 15.63°, 16.64°, 18.23°, 20.0°, and 22.1°; whereas the tri-clinic form of ART exhibited peaks at 2θ of 9.24°, 10.24°, 10.79°,11.58°, 14.56°, 14.95° and 18.01°. The original ART and the ART particlesfabricated by APSP without the polymers were of the orthorhombicform as seen from Fig. 2. The ART particles fabricated in the presenceof PVP or PEG, showed a prominent peak at 2θ=9.24°, which is alsofound in the triclinic form of ART. This indicated the formation of a tri-clinic formof ART in the presence of the polymers. The addition of a poly-mer affected the crystallization process and induced some changes in thecrystal structure of ART which resulted in the formation of the triclinicform. As previously reported, the conformation of both orthorhombicand triclinic molecules of ART and their bond lengths are quite similar,whereas the bond angles and the torsion angles are different for thetwo polymorphs [11]. It is predicted that the presence of the polymersmust have altered the bond angles and torsion angles which resultedin formation of the triclinic form of ART. Similar observations have

Fig. 2. X-ray diffractograms of the original ART and various ART samples prepared bythe APSP method.

been made in our previous studies where the triclinic form of ARTwas formed in the presence of carriers [21–23].

3.3. Thermal analysis by DSC

In order to understand the effect of the APSP process on the thermalproperties such as themelting temperature and themelting enthalpy ofART, DSC was conducted. DSC thermograms of the commercial ARTpowder is compared to the various samples prepared in Fig. 3. Theheat of fusion (ΔHf) values obtained from the DSC study are summa-rized in Table 3. The original ART powder used in this study had asharp melting endothermic peak at 156.4 °C. The endothermic meltingpeaks of the ART samples prepared by APSP were almost similar tothose of the original ART. However, the heat of fusion of the originalART powder (76 J/g)was higher than that of the ART particles prepared(59–66 J/g). Heat of fusion is proportional to the amount of crystallinityin the samples. These results suggest that the crystallinity of ART parti-cles was decreased when the particles were prepared by the APSP pro-cess, which was also supported by the XRD analysis. The endothermicmelting peaks of the ART particles prepared in the presence of PVP orPEG, lightly shifted to the lower temperature side (150 °C) and theheat of fusion (43–50 J/g) was also lower, indicating a lower crystallin-ity. This is also attributed to the formation of triclinic form of ART inthese samples as discussed above. The triclinic form is metastable com-pared to the thermodynamically stable orthorhombic form and hence,the melting temperature was lowered and heat of fusion was reducedfor these samples.

3.4. Dissolution study

The Multilinear Regression Model (data not shown) revealed thesignificant effects of the solvent to antisolvent ratio and stirringspeed on the dissolution of the drug particles prepared by APSP. Themodel predictions under the conditions of the experiments given inthe experimental design have been shown in Table 1. An “observedversus predicted” plot is displayed in Fig. 4. From this plot, the corre-lation coefficient of the model was found to be R2=0.945, suggestingthat the model is suitable for representing the factor—response rela-tionships within the variable ranges studied. Fig. 5 shows the predic-tion plots of dissolution against each of the studied factors such asdrug concentration, solvent to antisolvent ratio, stirring speed andflow rate. The optimal experimental conditions within the variableranges were identified as: drug concentration=15 mg/ml, solventto antisolvent ratio=1:20, stirring speed=1000 rpm and flow

Fig. 3. DSC thermograms of the commercial ART and various ART samples prepared bythe APSP method.

Table 3Dissolution parameters and the heat of fusion (ΔHf) of various ART samples.

Exp. no. RD4 h %DE4 h f1 f2 K R2 ΔHf (J/g)

Original ART 1 9.29 – – 0.0428 0.7501 76.21 2.69 23.17 146 40 0.1279 0.9375 65.82 2.31 18.95 102 46 0.1023 0.9619 66.63 2.76 23.71 152 39 0.1319 0.9423 65.24 2.53 21.08 125 43 0.1153 0.9531 66.15 3.22 29.23 210 32 0.1695 0.9164 63.66 3.26 29.10 209 32 0.1698 0.9320 63.27 3.83 34.70 270 27 0.2155 0.9284 61.58 3.89 35.70 281 26 0.2233 0.9148 61.09 2.45 20.07 114 44 0.1096 0.9631 66.410 2.31 18.81 101 47 0.1015 0.9625 66.711 2.66 22.49 140 40 0.1242 0.9496 65.312 2.55 21.14 126 43 0.1158 0.9531 66.213 3.49 31.79 237 30 0.1899 0.9181 62.114 3.42 30.71 226 31 0.1818 0.9246 62.615 4.05 37.15 297 25 0.2367 0.9100 60.716 4.06 38.06 306 25 0.2431 0.8885 60.317 3.19 28.37 201 33 0.1643 0.9314 64.018 2.91 25.36 169 37 0.1428 0.9349 64.919 2.64 22.26 137 41 0.1227 0.9478 65.620 2.86 24.74 163 37 0.1387 0.9371 65.021 2.12 16.89 81 51 0.0902 0.9703 67.022 3.71 33.44 256 28 0.2048 0.9302 61.823 2.30 18.01 93 48 0.0978 0.9767 67.124 3.13 27.38 190 34 0.1579 0.9431 64.225 3.00 26.30 179 35 0.1495 0.9361 64.7Low temp. 4.44 42.59 356 22 0.2891 0.8726 59.3ART (PVP) 4.89 46.88 403 19 0.3421 0.8827 43.9ART (PEG) 4.73 45.38 389 20 0.3220 0.8642 50.4

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rate=10 ml/min. The optimal dissolution was calculated to be54.24%. It can be easily noted from Fig. 5 that increasing the solventto antisolvent ratio, stirring speed and flow rate resulted in smallerparticle size and hence, greater dissolution rate. It was also observedthat a lower drug concentration gave better dissolution at smallervalues of stirring speed, solvent to antisolvent ratio and flow rate.However, at higher stirring speed, solvent to antisolvent ratio andflow rate; there was no significant difference in the dissolution ratebetween the samples produced at lower or higher drug concentra-tions. In other words, at the higher values of the three factors (stirringspeed, solvent to antisolvent ratio and flow rate), the drug concentra-tion did not have a significant influence on the dissolution of ART par-ticles. Following is the discussion about the experimental dissolutiondata, which is in agreement with the predicted data.

Fig. 6 shows the dissolution profile for the original ART powder andthe ART particles prepared by the APSP method. The dissolution of theoriginal ART powder in water was low with only about 13.10% of the

Fig. 4. Observed versus predicted dissolution with R2=0.945.

drug dissolving after 4 h as seen from Fig. 6 and Table 1. However, thedissolution of the ART samples 1 to 25 prepared by the APSP methodwas markedly increased as compared to the original ART powder asseen from the dissolution constant K values in Table 3. The dissolutionparameters such as RD (relative dissolution rate), %DE (percent dissolu-tion efficiency), f1 (difference factor) and f2 (similarity factor) are alsopresented in Table 3. Sample 16 showed the best dissolution amongthe ART samples prepared with the dissolution of 53.25% in 4 h. Com-pared to original ART powder, the ART particles prepared showed 2.12to 4.06 times enhancement in dissolution as concluded by the RD4 h

values in Table 3. The percent dissolution efficiency (%DE4 h) also in-creased significantly from 9.29 for the original ART to the highest valuesof 38.06 for sample 16.

The dissolution data for the APSP prepared samples presented agood fitting to the Noyes–Whitney equation as indicated by the R2

values of around 0.9 (shown in Table 3) except for the original ARTwhich showed a lower R2 value. The dissolution rate constant K calcu-lated from the Noyes–Whitney equation increased tremendouslyfrom 0.04 for original drug to 0.29 for the smallest ART particles pre-pared (Low temp). According to the Noyes–Whitney equation [7], thedissolution rate of a drug can be increased by reducing the particlesize to increase the particles surface area. The particle size of originalART powder was reduced by the APSP method, which improved thedissolution of the ART samples. The sample 16 showed the smallestparticle size and hence, the best dissolution among the 25 samplesprepared. The particle size was further reduced when the sample 16was prepared at the low temperature (the sample named ‘LowTemp’) giving even better dissolution. This indicates the dependenceof the dissolution properties of ART samples on particle size. In addi-tion, the extent of crystallinity also affects the dissolution of the drug.The crystallinity of the ART samples prepared by APSP was lower thanthe original ART powder, which also contributed to the increased dis-solution of the APSP samples.

Besides the particle size and crystallinity, the polymorphic form ofthe drug also affects its dissolution properties. In our study the triclin-ic form of ART particles was prepared in the presence of the polymer,PVP or PEG, and these samples presented the superior dissolution ratethan the orthorhombic form of the drug. It has also been previouslyreported that the orthorhombic form of ART possesses a greater den-sity and lower solubility in water than the triclinic form [11]. The dis-solution rate constant K for all the samples listed in Table 3 alsosupported the enhanced dissolution of the triclinic form of ART pre-pared in the presence of PVP or PEG, as compared to the remainingorthorhombic form of ART. The value of %DE4 h was enhanced from9.29 for the original ART to 46.88 and 45.38 for the ART precipitatedwith PVP and PEG, respectively. Being similar to the %DE4 h values,the RD4h values also showed a similar trend. Therefore, it is verifiedthat the precipitation of ART and hence, its dissolution can beinfluenced by the presence of a water-soluble polymer, PVP or PEG,in the APSP process.

Furthermore, comparison between the dissolution profiles of ARTparticles prepared from different formulations was made by the dif-ference factor (f1) and similarity factor (f2). The calculated f1 and f2values are reported in Table 3. f1 is zero when the test and drug refer-ence profiles are identical and increase proportionally with the dis-similarity between the two dissolution profiles. On the other hand,f2 is 100 when the test and reference profiles are identical and tendsto 0 as the dissimilarity increases. According to the data in Table 3,all the samples prepared by APSP method presented very high f1values and the f2 values were less than 50, indicating the dissimilarityof the dissolution profile of these samples with the original ART pow-der. The ART particles fabricated by APSP in the presence of PVP orPEG showed the smallest f2 values, and hence, the maximum dissim-ilarity with the original ART. These f1 and f2 values confirm the signif-icant improvement in the dissolution rates obtained for the ARTsamples after preparation by the APSP method.

Fig. 5. (a) Predicted dissolution versus drug concentration at the identified optimal conditions of solvent to antisolvent ratio=1:20, stirring speed=1000 rpm and flow rate=10 ml/min, (b) Predicted dissolution versus solvent to antisolvent ratio at the identified optimal conditions of drug concentration=15 mg/ml, stirring speed=1000 rpm andflow rate=10 ml/min, (c) Predicted dissolution versus stirring speed at the identified optimal conditions of drug concentration=15 mg/ml, solvent to antisolvent ratio=1:20and flow rate=10 ml/min, (d) Predicted dissolution versus flow rate at the identified optimal conditions of drug concentration=15 mg/ml, solvent to antisolvent ratio=1:20and stirring speed=1000 rpm. (♦) dissolution, (▲) lower confidence interval, (■) upper confidence interval.

475M. Kakran et al. / Powder Technology 237 (2013) 468–476

4. Conclusions

This study demonstrated that the APSP method is able to produceART particles with the significantly smaller particle size and thus, thehigher percent dissolution in water as compared to the original ART

Fig. 6. Dissolution profiles of original ART powder and APSP prepared samples.

powder. Various process parameters, such as drug concentration, sol-vent to antisolvent volume ratio, stirring speed, flow rate and temper-ature were investigated and optimized to produce the ART particlewith enhanced dissolution rates. The ART particles prepared by theAPSP method exhibited a lower crystallinity as revealed by the XRDand DSC analysis. The percent dissolution of ART particles dependedon particle size and crystallinity of the drug particles and the poly-morphic form of the drug. The commercial ART and ART particles pre-pared by APSP presented the orthorhombic form of the drug whereasthe ART particles formed in the presence of a polymer (PVP or PEG)had the triclinic form and showed the better dissolution than the or-thorhombic form. The APSP method has the advantages of usingwater as antisolvent and using a simple pumping, stirring and filter-ing system. The APSP method also gives the flexibility to producethe different polymorphs of the drugs and hence, adjust the size, mor-phology and properties of the drug particles produced. In addition tothat, APSP is very cost effective. To conclude, APSP represents an effi-cient method to obtain smaller drug particles with a higher rate ofdissolution in-vitro.

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