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American-Eurasian J. Agric. & Environ. Sci., 16 (2): 390-401, 2016ISSN 1818-6769© IDOSI Publications, 2016DOI: 10.5829/idosi.aejaes.2016.16.2.102171

Corresponding Author: Farhat Ullah, Department of Pharmacy, University of Malakand, Chakdara, Dir (L), KPK, Pakistan.Cell: +923339361513.

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Combinative Precipitation Ultrasonication Approach forFabrication for Stable Artemisinin Nanosuspension

Syed Muhammad Hassan Shah, Farhat Ullah, Shahzeb Khan,Syed Muhammad Mukarram Shah and Abdul Sadiq

Department of Pharmacy, University of Malakand, Chakdara, Dir (L), KPK, Pakistan

Abstract: The aim of this study was optimization of the processes and experimental conditions for fabricationof stable artemisinin (ART) nanosuspension using precipitation ultrasonication approach with subsequentenhanced dissolution rate compared to raw and micronized ART. The effect of important parameters includingconcentrations of stabilizer solution using polyvinyl alcohol (PVA), ultrasonication power inputs, length ofultra-sonication and effect of temperature were systematically investigated for fabrication of stable ARTnanosuspension. The optimized parameters to produce ART nanocrystals with smaller particle sizes were foundto be 0.15% of PVA, at 200 Watt ultrasonic power input over 15 minutes of ultra-sonication at 4°C temperature.The average particle size and polydespersity index for ART nano suspension were found to be 98.77± 1.5 nmand 0.186± 0.01 respectively. Crystallinity of the processed ART particles was confirmed using DSC and PXRD.Physical stability studies conducted for 30 days at different storage temperatures which include 4, 25 and 40°Cdemonstrated that nanosuspensions stored at 4°C and 25°C were most stable compared to the samples storedat 40°C. The ART nanosuspension showed significantly (P<0.05) increased (87 fold) in dissolution rate of theproduced nanocrystals compared to the micronized and raw ART which include 97%, 70% and 10.5%respectively after 60 minutes.

Key words: Artemisinin Optimization Experimental conditions Ultrasonic inputs Polyvinyl alcohol

INTRODUCTION suitable solvent followed by rapid injection into anti-

The recent literature has shown that more than 40% particles [10]. Aqueous solution containing stabilizers/of active pharmaceutical ingredients (APIs) coming from growth inhibitors is commonly used as anti-solventhigh throughput screening have tendency to suffer from phase. For a number of stable nano suspensionspoor aqueous solubility with subsequent erratic methylcellulose, hydroxypropylmethylcellulose, sodiumdissolution rate and bioavailability [1]. These drug lauryl sulphate, sodium deoxycholate, pluronics, tween-80compounds are very challenging to formulate through and polyvinyl alcohol have been reported the mostconventional approaches [2]. To address the issue of suitable stabilizers [11-12].poor water solubility multiple approaches are used which Nanoparticles could be produced by simple anti-include liposomes, salt, micells, neosomes [3], solid solvent nanoprecipitation method, which is a low energydispersion [4], micronisation [5], complexation [6], hot melt method and does not need highly sophisticatedextrusion [7] and nano suspensions [8]. Nanosuspensions technology. This approach has now become the focus ofof pharmaceutical active ingredients where the particles pharmaceutical scientists, however some limitations andhave increased surface area, high diffusion pressure and issues associated with this method are still need to bedissolution pressure have been reported the most addressed [13]. The technical challenge exploiting simplepromising approach [9]. precipitation approach is fabrication and stabilization

Solvent anti-solvent precipitation is an effective and to maintain particle size and particle size distributionway for fabrication of nano or micro-size drug crystals. (PSD). Nanoparticles are more prone to aggregation andBriefly, in this technique the drug is dissolved in a Ostwald ripening with more potential for sedimentation

solvent phase resulting immediate precipitation of drug

Am-Euras. J. Agric. & Environ. Sci., 16 (2): 390-401, 2016

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and caking. The stability of suspensions relies on the artemisinin and its derivatives which includemethod of generating particle and associated formulation dihydroartemisinin, artesinuate, artemether andcomponents [8]. Currently static mixer and impinging lumefantrine in combination with artemether [23], havereactors have been reported to control particle sizes and been reported strong antimalarial drugs which areparticle size distribution [14]. Also S. Khan et al. [8] has effectively used for 20 years [24]. Contrary toreported the controlled crystallization method while using conventional antimalarial drugs which are required 2-3different APIs (active pharmaceutical ingredients) which days to treat the malaria, the artemisinin act faster with andemonstrated that the stable nanosuspensions could be approximate parasite- and fever-clearance time of 32 h [25].produced if the process and experimental conditions are Nonetheless artemisinin is poorly water solubleproperly controlled. How do the nucleation and seed which causes the erratic absorption and poorcrystal control the particle growth in crystallisation bioavailability with consequent less antimalarial effect.process? has recently been investigated by combined It is therefore still an important issue to address the poorexperimental and simulation studies [15] and using water solubility issue of this drug which can potentiallyimpinging reactor as well [16]. boost up the dissolution and bioavailability. There has

In nanoprecipitation nucleation is very paramount to been a reported nanoparticles of the artemisinin usingbe controlled for tailoring particle sizes and their stirring method and evaporation precipitation [26].distribution [17] numbers of factors are responsible for However, the particle size was not effectively reducedcontrolling nucleation including supersaturation level, which caused stability issue as well. Moreover due tomixing time, micromixing and mass transfer etc [18]. ductile nature of this drug which is more prone to deformInsufficient micromixing in simple stirring process during rather than fragmentation at nano size [27], the wet millingmixings of solvent anti-solvent phases could potentially methods can blunt the effectiveness of this drug.be one of the major causes for instability of the produced The aims of the current study were to optimize bothnano suspensions[8]. Insufficient micromixing can reduce experimental (concentration of polymer) and processthe supersaturation level with subsequent slower conditions (temperature and ultrasonic inputs) fornucleation rate and broader size distribution [14]. To fabrication of stable artemisinin (ART) nanosuspensionsaddress the issue of micromixing in particular combined using modified and less expensive combined precipitationprecipitation-ultra-sonication has been very effective [19]. ultrasonication approach. Additionally the comparativeIrradiation has been known to be effective for mixing, to dissolution study was conducted to compare dissolutionaccelerate molecular diffusion and have the immense rate of the produced nanocrystals with micronized andintensity for mass transfer [18]. raw artemisinin to demonstrate the effectiveness of

In Western Medicine Malaria was one of the crystallized artemisinin. challenging diseases until the middle of the 20th century.At that time, malaria was still endemic in different parts of MATERIALS AND METHODSEurope and North America [20].

Malaria is caused by the genus Plasmodium. There Artemisinin (ART) was gifted by institute of Materialare four different species of plasmodium which cause Medica, China, supplier Chengduwagoot, Pharmaceuticalmalaria in human beings including P. ovale (malaria Co, Ltd having Batch No: (110916), polyvinyl alcoholquartana), P. falciparum (malaria tropica), P. malariae (PVA, 87-90% hydrolyzed and M. Wt 30, 000-70, 000) was(malaria tertiana) and P. vivax (malaria tertiana) [21]. purchased from Sigma Aldrich, (USA) having batch No:The first rationale attempt to treat malaria by Cinchona (MKBR5960V), dimethylformamide (DMF) Batch Nobark was made in early 18th century which has already 1373199, were purchase from Sigma Aldrich, (USA).been used in early 17th century in the treatment of fever Artemisinin nanosuspension was fabricated using[20]. Pyremethamine, choloroquine, halofantrine and combined precipitation ultrasonication method. Briefly, onmefloquine are the well established drugs which have the basis of artemisinin solubility 10mg/ml was dissolvedbeen used to treat malaria. These drugs are however in dimethylformamide (DMF) and a series of anti-solventoffered strong resistance by the malarial parasites. A new (aqueous) solutions containing different concentration ofclass of anti malarial group of drugs was derived from PVA(0.1%, 0.15%, 0.25%, 0.5% and 1%) were prepared.traditional Chinese medicine. The use of Artemisia annua Both organic (Solvent) and aqueous (Anti-solvent)has been reported for the treatment of haemorrhoids solutions were filtered through 0.4 µm filter to ensureand fever in China for at least 2000 years [22]. Currently maximum clarity. Afterwards, 02 ml of the solvent


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containing drug was injected with stirring at 1200rpm prepared by depositing a single drop of artemisinin nanointo pre cooled(4°C) anti-solvent solution (20 ml). The suspension onto a copper grid of 200 mesh coated withresulted precipitated artemisinin suspension was carbon which was dried at room temperature. The grid wasfollowed by ultrasonication at different ultrasonic input then negatively stained with 2% magnesium uranyl(50, 100, 150, 200, 250 and 300 Watt) at different time of acetate solution followed by drying at ambientinterval (5, 10, 15 and 20 min) using anti-solvent phase. temperature. The images were taken at 120KV.A number of Nano suspensions were prepared at millilitrescale (20ml) in order to identify the most suitable Differential Scanning Calorimetry (DSC): The meltingconcentration of the stabilizer solution. The time of point and heat of fusion of processed and unprocessedultra-sonication burst was set to 3 sec with a pause of artemesinin was determined using DSC (TA Instrument,3sec between two ultra-sonication burst. During Q500 and Q200, West Sussex, U.K) at heating range fromfabrication the temperature of the vial was controlled 25°C to 200°C at the heating rate 5°C/minute. Thewith ice cold water. After ultra-sonication at above experiment was performed under atmosphere of drymentioned parameters, the particle size measurements nitrogen having flow rate of 25 ml/min. All samples werewere carried out using Zetasizer Nano-ZS instrument analysed in triplicate.(Malvern instrument Ltd, UK).

Once the most appropriate concentration of Powder X-Ray Diffraction Studies (PXRD): Thestabilizer and ultrasonic power were identified, the effect crystallinity of raw and processed artemisinin particlesof anti-solvent phase temperature (04, 25 and 40°C) was previously recovered by centrifugation 16000 rpm wasalso evaluated for particle sizes. The physical stability determined by PXRD with powder diffractometerstudy on the produced nano suspensions was conducted (Bruker-Germany). Silicon-well sample holder wasfor a period of one month at three different temperatures employed for nanocrystals, while plastic sample holderwhich include 4, 25 and 40°C. was used for raw drug material. In triplicate the sample

The artemisinin nano suspension was subjected to and the raw drug was scanned in the range of 5-50° atcentrifugation at 16000 rpm for 01hr, the supernatant was 2 /min by using copper K as a radiation source withdecanted and the sediment nano crystals were collected 1mm slit at 1.542 Å wavelength.and characterized using differential techniques. Finally thecomparative in-vitro dissolution study of artemisinin Stability Studies: To assess the rate of crystals growthnano suspension was compared with raw and micronized by Ostwald ripening and aggregation the stability wasartemisinin powder. monitored for one month at different temperature i.e. (4, 25

Characterization days).Particle Size Measurement: The particle sizemeasurement of the produced nanocrystals was carried Dissolution Study: Dissolution rate of the producedout using Malvern Zetasizer Nano-Zs dynamic light nanocrystals was compared with raw and micronizedscattering instrument (Malvern Instruments, UK). artemisinin by using USP, dissolution type II apparatus at

Morphological Studies the drug sample were added separately to the dissolutionScanning Electron Microscopy (SEM): The morphology apparatus, maintaining the temperature of dissolutionof unprocessed (raw) artemisinin drug substance was medium at 37± 3°C and 5ml of the aliquots was withdrawndetermined at different ranges of magnification power by at specific interval of time (0, 2, 6, 10, 15, 30, 45 and 60using Scanning electron microscope (Quanta 400 SEM, min). All the samples were analyzed with reverse phaseFEI company Cambridge U.K). Preparation of samples was high performance liquid chromatography (RP-HPLC). involved fixing of artemisinin into a metal stub with thehelp of double sided adhesive tape. Statistical Analysis: Statistically data were interpreted

Transmission Electron Microscopy (TEM): The particle triplicate and results were given as mean± standard meansize of artemisinin nanocrystals was confirmed by TEM error (SEM). Mean values were compared using Anova-using transmission electron microscope (JEM-1200EX, test and difference were considered statisticallyElectron optics Lab, corp, Japan). The sample was significant at level (p < 0.05).

and 40°C) at regular time interval (0, 10, 15, 20, 25 and 30

200 rpm using reported method of kakran et al. [26]. All

using Statistics 8.1 software. All the data were run in


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RESULTS with increasing processing time (0-15 minutes) there was

Results Both the parameters were however increased at > 15Effect of Stabilizer Concentrations on Drug Particle minutes processing time (Fig. 1B).Size and Distribution: The average particle sizes andpolydespersityindex (PI) values of the artemisinin Effect of Anti-Solvent Phase Temperature on Particlenano suspensions produced by simple stirring and Size and Distributions: At optimum formulationcombined approach (Simple stirring and ultra-sonication) conditions previously identified for fabrication of ARTusing different concentrations of PVA solution nano suspensions, the anti-solvent solution (PVA(Anti-solvent) (0.1%, 0.15%, 0.25%, 0.5% and 1%) pre- solution) which was previously stored at different rangescold at 4°C, are shown in Table 1. The results of temperature (04, 25 and 40°C) showed marked effect ondemonstrated a substantial decrease in the particle the precipitation process with subsequent impact onsizes (98.77nm) and PI (0.18) values using the particle size and PI values (Fig. 2). There was observedcombined approach compared to the simple stirring that both particle size and PI are temperature dependent.method which resulted the particle size and PI values The minimum particle size (98.0) and PI (0.18) values were580.5 and 0.52 respectively at 0.15% PVA concentration obtained at 4°C (Fig. 2).(Table 1).

Effect of Ultrasonication Power Inputs and Length of nanosuspensions prepared at optimised process andTime: When the stabilizer concentration was fixed at formulation conditions was monitored at different storage0.15%, different Ultra-sonication inputs were applied temperature (04, 25 and 40°C) for one month. The particle(50, 100, 150, 200, 250 and 300Watt) for (05, 10, 15 and 20 size measurements and optical observations were carriedmin). The initial decrease in particle size and distribution out at 0, 10, 15, 20, 25 and 30 day. This study showedwas observed by increasing the power input. The most that ART nanosuspension was most stable at 4°C withstable nano suspension with minimum particle size was negligible particle growth (20 nm) and slight growthachieved at ultrasonic input at 200watt, with processing observed at 25°C (50 nm) (Fig. 3 A and B). Similarly at 4°Ctime of 15minutes (Fig. 1A). The increase in particle size and 25°C the increase in PI values was not substantialand distribution with rapid crystal size growth was (0.005 and 0.088). However a noticeable increase inhowever observed for ART by increasing the particle size (300 nm) and PI (0.45) occurred in theultrasonication inputs >200 watt. Whereas at 200 Watt nano-suspensions at 40 °C as shown in Fig. 3C.

observed decrease in the particle size and distribution.

Physical Stability: The physical stability of ART

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Table 1: Mean particle size of artemisinin(PS) in nanometre (nm) scale and distribution at different concentration of PVA (4°C) by simple precipitation andultra-sonication (Values are expressed as mean ± SEM)

Method Parameter PVA 0.1% PVA 0.15% PVA 0.25% PVA 0.5% PVA 1%

Simple Precipitation PS (nm) 912 ± 2.05 580.5 ±1.03 1213 ± 2.17 1636 ±1.70 2781 ±2.46PDI 0.764 ± 0.01 0.526 ±0.02 0.889±0.007513 0.926 ±0.02031 01 ±0.0

Precipitation Ultra-sonication PS (nm) 386.5 ±2.01 98.77 ±1.05 211 ± 1.26 1264.3 ± 2.37 1994.7 ± 1.86PDI 0.251 ±0.02 0.186±0.01 0.447 ±0.04007 0.744 ±00653 0.886±0.03032

Table 2: Dissolution rate of raw, micronized and artemisinin nanosuspensions as a function of different time intervals. Values in each column are significantlydifferent (P 0.05)

Dissolution time (minutes) Raw ART Micronized ART Nanosuspension ART

Mean ± SEM (LC 95% upper/ lower limits)

0 0 0 02 2 ±0.057 (2.24/1.75) 15 ±0.10 (15.43/14.57) 70 ±0.321 (71.38/68.61)c b a

5 3.066 ±0.176 (3.82/2.30) 22.06 ±0.145 (22.69/21.44) 70.26 ±0.384 (71.92/68.61)c b c

10 5.25 ±0.011 (5.29/5.20) 35 ±0.251 (36.08/33.91) 85.03 ±0.176 (85.79/84.27)c b a

15 7.50 ±0.015 (7.56/7.43) 55 ±0.230 (55.99/54.00) 90 ±0.100 (90.43/89.57)c b a

30 8.25 ±0.011 (8.29/8.20) 68 ±0.300 (69.29/66.70) 92.03 ±0.176 (92.79/91.27)c b a

45 9.03 ±0.145 (9.65/8.40) 70 ±0.404 (71.73/68.26) 92.66 ±0.260 (93±.78/91.54)c b a

60 10.250 ±0.005 (10.27/10.22) 72 ±0.208 (72.89/71.10) 97 ±0.115 (97.49/96.50)c b a


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Fig. 1: Effect of ultrasonication power input (A) and length of time (B) on mean particle size and distribution ofartemisinin nano suspension

Fig. 2: Effect of stabilizer (PVA) temperature on mean particle size and distribution (PDI) of artemisinin nano suspension


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Fig. 3: Effect of storage temperatures including; (A)4°C, (B) 25°C and (C) 40°C, on average Particle size andpolydespersityindex values of artemisinin nanosuspensions for 30 days

Fig. 4: SEM image of raw artemisinin


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Fig. 5: TEM image of artemisinin nanocrystals

Fig. 6: DSC profile of processed and unprocessed artemisinin particles

Fig. 7: PXRD patterns of raw and artemisinin nanocrystals

SEM Studies: Samples of raw Artemisinin were analysed TEM Studies: Transmission electron microscope (TEM)by scanning electron microscope at 200X magnification images of raw ART were taken at 80K and the averagepower, as shown in (Fig. 4) the artemisinin crystals were particle size was found to be bellow 100nm. As illustratedfound to be triclinic with average size of 200 micron. in (Fig. 5).


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Fig. 8: Comparative dissolution profile of raw, micronized and artemisinin nanosuspensions

DSC AND P-XRD Studies: Artemisinin nano crystals effective and simple. The appropriate selection andwere characterized by using DCS and P-XRD, which were concentration of the growth stabilizer/inhibitor is anidentified to be crystalline in nature. A single sharp important parameter for crystal size growth andendotherm was observed for both the nanocrystals and distribution. The growth of the crystals was inhibited byraw ART having slight reduction in the melting onset adsorbed stabilizer and for full coverage the concentrationtemperature (T and in the melting peak maximum, of the of the stabilizers should be enough for complete coverageonset)

nanocrystals compared to raw ART. Also dropped in heat on the crystals surface to provide sufficient sterric forceof fusion for nanocrystals was observed (Fig. 6). In PXRD of repulsion between the nanocrystals [13]. Owing tostudies both nanocrystals and raw artemisinin produced different crystal faces of nanocrystals the molecularsharp x-ray chromatograms which confirmed the interaction of drug nanocrystals and polymers is worthcrystalline nature of the produced nanocrystals (Fig. 7). mentioning, because it does not necessary that the same

Dissolution Studies: The dissolution rate of artemisinin connection, our findings showed that < 0.15% PVAnanosuspension was compared with unprocessed and concentration the growth in nanocrystals occurredmicronized (02 µm) artemisinin. The dissolution rate of rapidly. However at optimised PVA concentration (0.15%)artemisinin nano suspension was significantly faster as the nanosuspension was found stable with no particlecompared to its raw and micronized drug (Fig. 8). growth which demonstrated the sufficient adsorptionArtemisinin nanosuspension showed enhanced of polymer onto the produced nanocrystals surfacesdissolution release rate 70% while micronized and (Table 1). The effect of stabiliser concentration was alsounprocessed artemisinin could only release 15 and 2% validated by both simple stirring and combineddrug respectively within 2 min. For artemisinin approaches where in both methods we observed thenanosuspension statistics showed (Table 2) significance stable nano suspensions at 0.15% PVA concentration.(p< 0.05) difference in the in-vitro studies as compared to Owing to high level micro mixing and mass transfer, theraw and micronized drugs. combined approach was found more effective in terms of

DISCUSSION crystals growth could result due to insufficient coverage

Effect of Stabilizer Concentrations on Drug Particle Size the stabilizer hinder transmission of ultrasonic vibration,and Distribution: The approach utilized here for due to high viscosity of solution, which adversely affectfabrication of artemisinin nanosuspension was found cost the diffusion between solvent and anti-solvent [19].

type of polymers works for different drugs [28]. In this

particle size reduction and PI [29]. Agglomeration and

of the crystals by stabilizer, while high concentration of


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Effect of Ultra-Sonication Power Inputs and Length of Physical Stability: The physical stability study of ARTTime: In optimisation of ultrasonication input power and nano suspensions at different temperature waslength of processing time a range of input powers (50-300 considered very paramount to investigate the impact ofwatt) and processing time including 5 to 20 minutes were temperature on nanocrystals growth. This study revealedevaluated for production of stable nanosuspensions that ART nano suspension was very stable at 04 and 25°C(Fig. 1). Our findings suggested that stable with little increased in size after end of day 10 , with nonanosuspension was obtained at ultra-sonication input marked change in particle size and distribution over one(200 Watt) with processing time of 15 minutes. month. Although, at 40°C increased in particle size (300.0Nonetheless increase in ultra-sonication input >200 Watt nm) and its distribution was observed (Fig. 3 C).and processing time >15 minutes resulted increase in Probably, at high temperature the growth of theparticle size and PI with rapid crystal growth. This particle size is due to Ostwald ripening [33]. At lowincrease in particle size can probably be caused by temperature the kinetic energy and diffusion are likely toincrease in temperature due to high energy input [19]. be decreased, which is expected to increase the level ofSimilarly the longer time was not effectively helped to supersaturation and rapid nucleation, since the number ofreduce the particle size which might be due to the micro nuclei increased with decreased of solute on each nucleimixing level which has already been achieved at 15 [32].minutes. The stability was interestingly increased afterfinding the most appropriate concentration of stabilizer Morphology Studies: Morphology of the producedsolution (0.15%) and ultrasonic inputs 200 Watt for nanoparticles has a direct impact on the dissolution(15minutes) (Fig. 1B), which is due high power of micro studies and other physicochemical attributes. It ismixing [18]. The energy addition step (ultrasonication) therefore very imperative to find whether the producedwas found to be a kind annealing (conversion of particles are amorphous or crystalline in nature. Inthermodynamically unstable matter to sable one) step for contrast to SEM images of the raw ART which showedstable nano suspension by lowering its energy. The the particle size around 200 micron and shape was tricliniclowering of energy can be achieved by converting less (Fig. 4), the TEM analysis demonstrated that the particleordered solid state to more order form i.e., from size substantially reduced to nano level (Fig. 5). Theamorphous form to crystalline state and vice versa or nanocrystals were found separate from each other withby reordering the stabilizer/growth inhibitor at the mix spherical and crystalline shapes. The surfaces weresolid-liquid, or increasing adsorption of surfactant on the found very rough as compared to the raw ART. TEMsurface of crystal, which interns will reduce the surface images showed that particle size of artemisinin was in thefree energy [30]. nanometre scale that is being consistent with dynamic

Effect of Anti-Solvent Phase Temperature on Particle size was bit smaller compared to the DLS which is becauseSize and Distributions: Figure 2 illustrated that increasing of the difference in measuring principles of thethe temperature of stabilizer (PVA) solution from (04-40°C) techniques. In DLS the hydrodynamic layer around theresulted in nanocrystals growth with subsequent particles is also measured, while in TEM the particles areunstabilisation of the produced nanosuspensions. Lower directly measured [34].the temperature of the solution (04°C), smaller the particlesize and distribution was observed. While increased in DSC AND P-XRD Studies: DSC and PXRD studies havemean particle size and its distribution was observed with been reported very important to identify the changes insudden growth of crystals at high temperature resulting packing density and crystallinity of the producedin unstable nano suspension. It has been reported that in nanoparticles [35]. During nanoparticles formationsolvent and anti solvent precipitation method the processes including wet milling and anti-solventtemperature of stabiliser solution (anti solvent) have crystallisation either the raw crystals are broken down duegreater impact on produced nanocrystals [31]. At high to attrition forces or new particles are formed fromtemperature the solubility of the ART increased, therefore solution. The newly produced particles should behaveupon mixing decreasing the level of supersaturation, differently from the unprocessed particles. In thiswhich resulting the low rate of nucleation having less connection DSC and PXRD studies are very helpful toavailability for nuclei for the growth of crystals, ultimately compare and contrast the melting point, heat of fusionresulting large crystals [32]. and crystallinity of unprocessed and processed particles.

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light scattering (DLS) determination. However the average


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DSC analysis exhibited that melting point and heat of preparation of stable nanocrystals with smaller particlefusion of produced ART nanoparticles were slightly sizes. This study also concludes that pertaining toreduced (Fig. 6). This decreased in heat of fusion is due to chemical engineering aspects if both process andthe adsorbed stabilizer(s) on the nanocrystals surface in formulation conditions are properly controlled then thistrace amount [36, 37]. Also this could be because of the economic technology could be scaled up for nanolow packing density in case of nanoparticles [35]. formulation of this challenging API (ART).

In PXRD studies there was observed that intensity ofthe produced nanoparticles reduced, broadened and ACKNOWLEDGMENTSdisappearance of some peaks also occurred (Fig. 7). Dueto particle size broadening effect, the peak for ART was I would like to acknowledge the financial supportslightly broadened, which is the key to attain low particle received from the International Research and Initiativesize. There was observed disappearance and broadening Programme (IRSIP) of Higher Education Commission ofof some of the peaks for ART nanocrystals which is Pakistan (HEC) and School of Pharmacy, Institute of Lifeconsidered to be due to preferred effects of orientation. Sciences Research, University of Bradford, West

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