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Consideration of the solid state for resveratrol nanocrystal production
Accepted Manuscript Consideration of the solid state for resveratrol nanocrystal production
Tao Liu, Rainer H. Müller, Jan P. Möschwitzer PII: DOI: Reference:
S0032-5910(18)30220-1 doi:10.1016/j.powtec.2018.03.028 PTEC 13259
To appear in:
Powder Technology
Received date: Revised date: Accepted date:
6 July 2017 18 February 2018 15 March 2018
Please cite this article as: Tao Liu, Rainer H. Müller, Jan P. Möschwitzer , Consideration of the solid state for resveratrol nanocrystal production. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ptec(2017), doi:10.1016/j.powtec.2018.03.028
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ACCEPTED MANUSCRIPT Consideration of the solid state for resveratrol nanocrystal production
Tao Liu 1; Rainer H. Müller 2; Jan P. Möschwitzer 2,3*
Dept. of Pharmaceutical Engineering, College of Chemical Engineering, Qingdao University of Science and
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1
Technology, Qingdao, China
Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin,
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2
Berlin, Germany
Advance Pharma GmbH and Steiner Deutsche Arzneimittel GmbH, Berlin, Germany
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*Corresponding author: Dr. Jan Peter Möschwitzer
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Tel.: +49-177-2306843
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E-mail address: [email protected]
ACCEPTED MANUSCRIPT Abstract
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Production of nanosuspension for drug bioavailability improvement has been accepted and frequently used in pharmaceutical industry. Normally, the preparation started from crystalline drug. However, some previous studies found that amorphous drug powder obtained from processes such as spray-drying might contribute to nanosizing process, i.e. reduced homogenization cycles and smaller achievable particle size. The mechanism behind this improved behavior was still unclear. In order to systematically investigate, three technologies, i.e. spray-drying, rotary evaporation and quench cooling, were used to modify drug powders, which were all processed by high pressure homogenization (HPH). Predominately amorphous solids have been obtained after processing both spray-drying (spherical particle) and rotary evaporation (no obvious morphology modification). However, those samples could not be beneficial to efficiency of nanosizing process. The smallest particle size of nanosuspensions was achieved by using spray-dried solid possessing predominately crystallinity (slightly reduced crystallinity) and changed morphology (more breakage points). Therefore, both the modification of morphology and solid state control were confirmed crucial for the followed HPH efficiency. In another part of this study, it was found that the directly dried nanosuspensions used in many previous studies were actually not suitable for a proper solid state analysis by differential scanning calorimetry (DSC). A separation step of drug and stabilizer/surfactant is strongly recommended in order to improve the validity of solid state measurement for drug nanosuspensions.
ACCEPTED MANUSCRIPT Fig. 1 Schematic description of processes for investigation.
Fig.2 DSC curves (top) and PXRD patterns (bottom) of spray-dried samples with different resveratol (RVT)/sodium cholate (SC) ratios and unmodified RVT. Fig. 3 DSC curves (top) and PXRD patterns (bottom) of rotary evaporation samples different
resveratrol
(RVT)/hypromellose
(HPMC)
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with
and
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unmodified RVT.
ratios
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Fig. 4 DSC curves (top) and PXRD patterns (bottom) of quench cooled resveratrol (RVT) and unmodified RVT.
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Fig. 5 SEM photos of unmodified resveratrol, spray-dried powders, rotary evaporation samples and quench cooled resveratrol (scale bar: 5 μm, magnification: 5000X). Resveratrol/sodium cholate ratios for spray-drying were 1:0 (A), 1:0.146 (B) and 1:0.854 (C). Resveratrol/HPMC ratios for rotary evaporation were 2:1 (D), 1:1 (E) and 1:2 (F).
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Fig. 6 Particle sizes (LD and PCS) of nanosuspensions produced from spray-dried resveratrol (RVT) with different sodium cholate (SC) ratios as well as unmodified RVT after 20 HPH cycles.
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Fig. 7 Particle sizes (LD and PCS) of nanosuspensions produced by using rotary evaporation resveratrol (RVT) with different HPMC ratios as well as
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unmodified RVT after 20 HPH cycles.
Fig. 8 Particle sizes (LD and PCS) of nanosuspensions produced by using quench cooled resveratrol (RVT) as well as unmodified RVT after 20 HPH cycles.
Fig. 9 DSC thermographs (top) and PXRD patterns (bottom) of unmodified resveratrol (A), physical mixture of resveratrol/sodium cholate in 1:1.1 (B), nanosuspension produced from unmodified resveratrol with sodium cholate (C) and sodium cholate was removed from nanosuspension (D).
ACCEPTED MANUSCRIPT 1 Introduction Bead milling and high pressure homogenization (HPH) are currently the two primary technologies for production of drug nanocrystals [1]. In addition, several combinative technologies, e.g. spray-drying [2] or freeze-drying [3] followed by HPH, have been developed. These combinative methods normally use a pre-treatment step such as
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spray-drying to modify the physical properties of drug powders prior to the top-down process. It was found that the pre-treated powders could significantly improve the
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nanosizing performance due to the reduced HPH cycles and much smaller achievable
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particle size compared to standard HPH technology [4]. For example, smaller glibenclamide nanocrystals (z-average, 236 nm) were produced by using the
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spray-dried powder compared to standard HPH (z-average, 772 nm) [2].
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A previous study showed that spray-dried drug with very low crystallinity was proven to be more effective for particle size reduction than its crystalline form [2]. This phenomenon led to the assumption that amorphous active pharmaceutical ingredient
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(API) might be beneficial for obtaining a smaller particle size and thus could improve
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the millability of the pre-treated API. However, in case of resveratrol nanocrystal production, it was difficult to build a correlation between the starting crystallinity and
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minimal achievable particle size after HPH [5]. In addition, all above related studies were only achieved by one process, i.e. spray-drying, however, amorphization can be
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achieved by various approaches based on different mechanism and consequently the obtained sample might possess different millability. Therefore, these conflicting results led to the necessity to understand the mechanism behind the improved performance systematically by comparing different pre-treatment technologies. In this regard the effect of amorphization is a key parameter to be investigated. Resveratrol is a poorly soluble plant compound, which possesses potential pharmaceutical effects. Its low bioavailability could be mainly attributed to the poor solubility and rapid metabolism [6]. Nanosizing has been proven to be an effective way to process it for medical application, such as dermal targeting of irritant contact dermatitis [7].
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Solid state modification of one compound by different methods was confirmed possible [8]. There are three rapid modification methods available, i.e. spray-drying, rotary evaporation and quench cooling, which are frequently used to modify drug
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physical properties, especially, for drug amorphization [9, 10]. These three methods constitute totally different procedures to generate amorphous APIs. Therefore,
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theoretically different kinds of modified APIs can be produced by applying those
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processes.
Spray-drying is a rapid drying technology widely used by the pharmaceutical industry.
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Drug properties e.g. particle morphology, particle size and solid state can be modified after the process [11, 12]. Spray-drying is an established technology for producing
drug
solubility
improvement.
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amorphous solid dispersions, which is a frequently used formulation technique for Various
polymers,
such
as
hydroxypropyl
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methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) have been used recently for
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the production of solid solutions [13]. In addition, carriers are not always essential for the formation of amorphous APIs. The rapid organic solvent evaporation may directly result in a modification of the solid state when spray-drying was applied [14]. As
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mentioned above, spray-drying has already been used successfully as the first step of
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the combinative particle size reduction method.
Flash evaporation by using a rotary evaporation is another way to achieve fast drying. The evaporation is achieved in a vacuum system, in which the boiling point of solvent is reduced and the corresponding drying process can be accelerated. This method can be also used to produce amorphous solid dispersion when proper drug to polymers ratios are used [15-17].
Quench cooling of a melt is another simple way for producing amorphous APIs. The molten drug is quench cooled by using liquid nitrogen. Irregularly arranged molecules
ACCEPTED MANUSCRIPT can be solidified when the cooling process is performed fast enough [14]. The process may also lead to chemical degradation because of the elevated temperatures applied [18]. However, the milling properties of those quench cooled samples are unknown as no corresponding literature has been found to date.
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In order to assess the impact of solid state changes on the particle size reduction efficiency, a proper solid state analysis is crucial for both, the unmodified and
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modified starting material, as well as for the drug nanocrystals obtained after the process. The latter characterization is important, as the solid state of drug
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nanoparticles can significantly affect the stability and dissolution rate of the nanosuspension [19]. A high energy process, such as milling, may induce a modified
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solid state of the processed material [9]. In another study, Sharma et al. have investigated the influences of HPH on the drug solid state after the process. No
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amorphous API or only a slight surface amorphization was found after the HPH
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process [20].
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To date, directly dried nanosuspensions were used in most publications to analyze the solid state of the drug nanocrystals. However, the drying process may have an influence on the solid state of API and lead to alteration as discussed above. In
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addition, the API might have solubility in the stabilizer/surfactants used for HPH. Therefore, it would be difficult to judge whether the amorphous fraction resulted from
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the milling or the downstream process [21].
The aim of this study was to investigate the resveratrol solid state throughout the nanosizing process. Spray-drying, flash evaporation by rotary evaporation and quench cooling were respectively used to modify API physical properties, i.e. solid state as well as particle morphology. All the modified powders were subsequently processed by HPH (Fig. 1). The particle sizes of nanosuspensions produced from those different starting solids were compared. In another part of this study, solid state of drug nanosuspension was carefully investigated. Two different measurement methods (with
ACCEPTED MANUSCRIPT and without stabilizer) were performed. Finally, an accurate measurement method of nanosuspension was expected.
2 Materials and methods
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2.1 Materials
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Resveratrol was obtained from E. Denk Feinchemie GmbH, Germany. Hydroxypropyl methylcellulose (HPMC) was purchased from Colorcon, England and sodium cholate
SC
from Sigma-Aldrich Chemie GmbH, Germany. Purified water was supplied from a Milli-Q system (Millipore GmbH, Germany). Cellulose nitrate membrane filters (0.1
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2.2 Resveratrol modification techniques
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μm, Ø 25mm) were purchased from Whatman GmbH, Germany.
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2.2.1 Spray-drying
Spray-drying of organic resveratrol solutions was performed by using a mini spray-dryer B-290 equipped with an inert loop B-295 (Büchi Labortechnik AG,
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Switzerland). Ethanolic solutions of resveratrol and sodium cholate in the ratios of 1:0, 1:0.146 and 1:0.854 were prepared. The following parameters of spray-drying were
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used throughout the study: inlet temperature 105oC, aspirator 80%, solution feed rate 10% and air flow 414 L/hour.
2.2.2 Rotary evaporation
Resveratrol and HPMC (2:1, 1:1 and 1:2) were dissolved in a mixture of water and ethanol (1:3). The obtained solutions were dried rapidly by a Büchi Rotavapor-R (Büchi Labortechnik AG, Switzerland) under a pressure of 100 mbar. The water bath temperature was set at 70oC and the rotary speed was set to 9.
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2.2.3 Quench cooling
Resveratrol was heated on a metal plate until the material was completely molten. Subsequently, the molten mass was immediately poured into liquid nitrogen. The
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quenched sample was collected and grinded manually in a mortar to produce
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micronized drug.
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2.3 High pressure homogenization (HPH)
A Micron Lab 40 homogenizer (APV Deutschland GmbH, Germany) equipped with a
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temperature control unit was used to produce resveratrol nanosuspensions. Firstly, the different spray-dried powders were dispersed in an aqueous sodium cholate solution
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with corresponding concentration to maintain a constant resveratrol/sodium cholate ratio of 1:1.1. Samples processed by rotary evaporation were suspended in different
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HPMC solutions to obtain suspensions with a ratio of 1:2 (resveratrol/HPMC). The
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milled quench cooled powder was suspended in an aqueous dispersion medium containing 1.1% w/w sodium cholate. The drug content of all suspensions to be nanosized by HPH was kept constant at 1.0% w/w. All coarse suspensions were first
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stirred for 1 minute by using an Ultra-Turrax T25 (IKA-Werke GmbH & Co. KG, Germany) at 8000 rpm. Subsequently, the obtained microsuspensions were
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pre-homogenized applying 2 cycles at 500 bar and 1 cycle at 1000 bar. 20 homogenization cycles at 1500 bar were employed to produce nanosuspensions at 5oC.
2.4 Differential scanning calorimetry (DSC)
The solid state of the unmodified resveratrol and all modified samples was analyzed by using a Mettler DSC 821e (Mettler Toledo, Germany). In addition, the solid state of resveratrol nanosuspension was also analyzed. The nanosuspension was dried for
ACCEPTED MANUSCRIPT measurement. Here, the resveratrol nanosuspension was handled in two different ways. One approach was to directly dry the nanosuspension to obtain powders which were subsequently analyzed by DSC. In another approach, the nanosuspension was filtered (0.1 μm) and the residual on filter membrane was washed by water in order to remove an excess of stabilizer. The drug nanoparticles without stabilizer were then collected as residuals on the membrane and then were dried. Finally, all dried samples were
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analyzed by DSC. A heating rate of 10oC/min and an accurately weighted sample
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amount of about 1-2 mg was used for all DSC measurements.
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2.5 Powder X-ray diffraction (PXRD)
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The solid state of unmodified resveratrol, modified powders and resveratrol nanosuspension were analyzed by PXRD. Measurement of nanosuspension in liquid
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state by adding viscosity enhancer (locust bean gum) in this study was found below the detection limit because of the low API concentration (no API characteristic peak
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in the pattern). Therefore, dried nanosuspension was also used for measurement. Both
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directly dried nanosuspension and stabilizer removed nanosuspension (i.e. filtered nanosuspension) were used for analysis (same sample preparation as mentioned in DSC measurement). The analysis was performed by using a Philips PW 1710
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diffractometer (Philips Industrial & Electro-Acoustic Systems Division, The
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Netherlands). The scanning angle was from 0.6º to 40º with steps of 0.02º per second.
2.6 Particle size analysis
2.6.1 Photon correlation spectroscopy (PCS)
The mean particle size (z-average) and the polydispersity index (PdI) of nanosuspensions were measured by PCS using a Zetasizer Nano ZS (Malvern Instruments, UK). Each nanosuspension of 50 µl was diluted in 3 ml purified water for 10 measurement runs in triplicate. The measurement temperature was set the same
ACCEPTED MANUSCRIPT as the sample temperature.
2.6.2 Laser diffractometry (LD)
A Mastersizer 2000 (Malvern Instruments, UK) was used to analyze the particle size
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distribution of all nanosuspensions after HPH. A real refractive index of 1.69 and an imaginary refractive index of 0.02 were used for the data calculation [22]. 3 runs were
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performed for each measurement. The obtained particle size is volume-based. LD diameter d (0.5) or d (0.9) means that 50% or 90% of the particles are smaller than the
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2.7 Scanning electron microscopy (SEM)
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given size.
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SEM was used in order to assess the particle morphology, i.e. the crystal habit of all starting materials, i.e. unmodified resveratrol, spray-dried resveratrol, rotary
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evaporation samples and quench cooled powder. A scanning electron microscope
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(Hitachi S-520, Japan) was used for the assessment. The powder was placed on a carbon holder and gold was coated with a layer thickness of 22 nm-25 nm prior to the
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analysis.
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2.8 Statistic Analysis
The statistical significance of differences between experimental groups was calculated by using Student’s t-test and p values <0.05 were considered significant.
3 Results and discussion 3.1 Effect of modification step on solid state of resveratrol As shown in Fig. 2, the unmodified resveratrol was crystalline according to both DSC and PXRD results. The corresponding DSC curve and PXRD pattern of spray-dried
ACCEPTED MANUSCRIPT resveratrol without sodium cholate were comparable to these of raw resveratrol. The spray-drying process could not alter the crystallinity of resveratrol, which means the dissolved API in ethanol tended to recrystallize during the spray-drying evaporation processes. A position shift of the melting peak was found in the DSC curve of the spray-dried sample with 1:0.146 of resveratrol/sodium cholate. However, this result
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was not matching with the PXRD patterns of the same sample, in which characteristic peaks of resveratrol could still be observed. It is assumed that resveratrol has some
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solubility in sodium cholate when the temperature was elevated during the DSC measurements. In addition, the melting point of resveratrol was decreased by sodium
SC
cholate during the heating period of DSC. A similar phenomenon was observed in other studies [20, 23]. A reduced crystallinity was found when a high amount of
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sodium cholate was used for the co-spray-drying. The DSC curve of spray-dried sample containing 1:0.854 of resveratrol/sodium cholate showed an exothermic glass
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transition peak and largely reduced melting peak. This indicated that predominately amorphous solid was achieved. The amorphous halo found in PXRD pattern of this
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sample also confirmed the DSC results. Therefore, only the spray-drying process
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could not change the crystallinity and it was essential to apply sufficient sodium cholate.
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The rotary evaporation process using HPMC as stabilizing polymer yielded solid dispersions as described in the literature[13]. It could be clearly seen from the DSC
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curves that the melting peak of resveratrol decreased as the HPMC content increased. No endothermic peak was observed in the DSC curve, when a 1:2 ratio of resveratrol/HPMC was processed (Fig. 3). This means that resveratrol molecular irregularly dispersed in the HPMC carrier and a fully amorphous solid dispersion was produced. In addition, all PXRD results were in agreement with the DSC measurements. These results confirmed that the drug to HPMC ratio had a significant influence on the solid state modification of resveratrol.
Quench cooling was also performed in order to modify the solid state of resveratrol.
ACCEPTED MANUSCRIPT Surprisingly, no reduced crystallinity was found by using this method as shown in Fig. 4. Both DSC and PXRD results of the quench cooled resveratrol were similar to these of the unmodified resveratrol.
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3.2 Particle morphology characterization of different modified powders
Fig. 5 shows the particle morphology of unmodified resveratrol and modified
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resveratrol powders yielded by the different modification processes. The spray-dried
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powders clearly showed different particle morphologies as compared to the unmodified resveratrol. A spherical structure was found for all three spray-dried
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samples. This means the droplets dissolved API during spray-drying possessed a spherical shape, which is normally the most stable shape for a droplet [24]. In addition,
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the spray-dried powders showed obvious a decrease of crystallite size. The rotary evaporation samples have also shown different particle morphologies compared to
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both the unmodified resveratrol as well as the spray-dried resveratrol. Although the
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morphology of resveratrol was totally modified after processing with a rotary evaporator, a very fragile structure could not be identified when assessing the SEM
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pictures.
The particle morphology of quench cooled sample is also shown in Fig. 5. Particle
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morphology of the quench cooled resveratrol was still similar to that of raw resveratrol with a compacted structure. Therefore, quench cooling was not a suitable process to modify the resveratrol particle morphology. 3.3 Particle sizes of nanosuspensions Comparing to the only spray-drying modification in the previous study [5], spray-drying and rotary evaporator processes produced different crystallinity of resveratrol (including the crystal sample from spray-drying without sodium cholate) by using different types (ionic and polymeric) and amounts of corresponding
ACCEPTED MANUSCRIPT excipients. In addition, both rotary evaporator and quench cooling led to different solid state as well as particle morphologies compared to the spray-dried powders and samples in previous study [5]. All the obtained samples were produced by HPH process. Fig. 6 shows the particle sizes of nanosuspensions obtained from spray-dried
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resveratrol and unmodified resveratrol after 20 homogenization cycles. The particle sizes of the predominately amorphous sample (1:0.854 resveratrol/sodium cholate)
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were 0.302 μm (d (0.5)), 1.665 μm (d (0.9)), 642 nm (z-average) and 0.306 (PdI) after
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20 homogenization cycles. These particle sizes were comparable to those yielded by the unmodified resveratrol. In contrast, the smallest particle size (d (0.5), 0.242 μm; d
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(0.9), 0.801 μm; Z-average, 356 nm; PdI, 0.113) was achieved by using predominately crystalline spray-dried sample (1:0.146) (p<0.05). The particle size of spray-dried
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resveratrol without sodium cholate was also smaller compared to the result of a nanosuspension generated with unmodified resveratrol after 20 homogenization
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cycles (p<0.05), although the solid state was similar to unmodified resveratrol.
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Therefore, it confirmed the necessity of morphological improvement for an effective particle size reduction during HPH process, which could not be clearly answered in the former related study [5].
It could be possible that the improved performance of
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this spray-dried sample might result from the increase of crystal weak points or decrease of crystallite size as shown in SEM photos. Although those three spray-dried
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powders showed comparable morphology, the one with lowest crystallinity could not be beneficial for achieving an improved particle size reduction effectiveness and one possible reason was that the recrystallization happened during the HPH process.
Fig. 7 shows the particle sizes of resveratrol nanosuspensions from the rotary evaporation samples compared to unmodified resveratrol. The obtained mean particle sizes (z-average) after 20 homogenization cycles by using solid dispersion of resveratrol/HPMC (1:2, 1:1 and 2:1) were 559 nm (PdI, 0.148), 558 nm (PdI, 0.152), and 520 nm (PdI, 0.136), respectively. These results were comparable to the particle
ACCEPTED MANUSCRIPT size of unmodified resveratrol nanosuspension (579 nm) (p>0.05). The LD results also confirmed that all particle sizes of modified samples were similar to the unmodified one. As shown in Fig. 7 in general the LD d (0.9) value of rotary evaporation samples were smaller than that of the unmodified resveratrol, but d (0.5) values became larger. The generation of amorphous solid dispersions by using
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additional polymer (HPMC) could not contribute to the obtained average nanoparticle size according to both PCS and LD. Those results from rotary evaporation samples
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showed that the amorphization method and added excipient could be also crucial for drug modification. Although crystallinity at different levels was achieved by rotary
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evaporation with polymer (HPMC), it could not influence the minimal achievable
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particle size positively.
The particle size of quench cooled resveratrol after 20 homogenization cycles was
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comparable to that of unmodified resveratrol nanosuspension according to both LD and PCS measurements (p>0.05) (Fig. 8). When comparing this sample and the
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spray-dried sample without sodium cholate, the solid state of both were similar,
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however, the particle morphologies were found to be totally different. In addition, some recent studies found that the particle morphology of starting drug powder was an important parameter of the nanocrystals production [3, 25]. Therefore, it can be
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concluded that the modification of the resveratrol morphology played an important
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role for the subsequent HPH process.
According to the process performance when processing these three kinds of modified powders, only spray-dried samples contributed to an improved process efficiency compared to unmodified resveratrol. The modification of solid state by using rotary evaporation could not be directly correlated with the final nanoparticle size. Therefore, it is also proved that a valid modification of resveratrol for the followed HPH was highly dependent on the amorphization method and applied co-processed excipient. The modified morphology of spay-dried powder was also an important aspect for achieving a smaller particle size. In order to obtain the smallest particle in
ACCEPTED MANUSCRIPT nanosuspension, the powder used for HPH was expected to possess modified morphology (spray-dried, more breakage points) as well as predominantly crystal solid state (slightly reduced crystallinity but not amorphous).
3.4 Characterization of resveratrol solid state in nanosuspension
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Conflicting data have been found with regard to the solid state of the resveratrol nanocrytals after the HPH process. An exothermic peak and a very small melting peak
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were found in DSC thermogram (C in Fig. 9) of unmodified resveratrol
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nanosuspension. This would normally indicate a solid state change towards a predominately amorphous solid. However, the characteristic peaks in PXRD
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diffractograms (C in Fig. 9) indicated a predominately crystalline state of this particular sample. As the directly dried nanosuspension contained resveratrol and
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sodium cholate simultaneously, it was hypothesized that the presence of sodium cholate could have led to incorrect DSC results.
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In order to test this hypothesis, sodium cholate was removed to a great extend by
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filtration to exclude any potential risk of an alteration of the solid state of resveratrol during the DSC heating. The results for this study are shown in Fig. 9 (D). The filtrated sample only showed one melting peak and a slight reduction of melting point
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(266oC) which might be attributed to the smaller particle size [26]. The DSC and
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PXRD results were in good agreement when the filtrated samples have been analyzed. Additionally, a physical mixture of resveratrol and sodium cholate (1:1.1) was investigated (B in Fig. 9). The PXRD pattern of the nanosuspension was comparable to the physical mixture of resveratrol and sodium cholate. Two melting peaks (108oC and 243oC) were found in the DSC thermograph. The melting point of resveratrol (243oC) was lower than that of unmodified resveratrol. This reduction of melting point has also confirmed the solubility of resveratrol in sodium cholate during the DSC heating period. The lower melting temperature of the dried nanosuspenison compared to the physical mixture might be again a result of the smaller particle size
ACCEPTED MANUSCRIPT of resveratrol. Here it can be assumed that the solubility of resveratrol in the stabilizer, i.e. sodium cholate, was further improved due to the smaller particle size of resveratrol.
The phenomenon shown above confirmed that, if the nanosuspension cannot be
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measured in liquid state due to the low API concentration, the directly dried nanosuspensions used in most previous studies were actually not suitable for a proper
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solid state analysis by DSC. If the API has the solubility in the specific surfactant or polymer used for the physical stabilization of the particles, a kind of solid dispersion
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can be generated during the heating period of the DSC measurement. The real solid state of an API could be masked by using this analytical procedure. This can result in
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misleading conclusions. This problem about DSC measurement of nanosuspension was firstly proposed in this study, which has not been aware in most related
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nanocrystal publications. Therefore, it is strongly recommended to remove an excess of surfactant, stabilizer and polymer from the nanosuspension by filtration or other
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purification methods before DSC measurement.
4 Conclusion
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The resveratrol powders modified by spray-drying, rotary evaporation and quench cooling were nanosized by HPH, respectively. Spray-dried powders containing tiny
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amounts of sodium cholate or without sodium cholate showed predominately crystalline solid state. Using these samples, the HPH process was more efficient compared to all other modified or unmodified starting materials. Predominately amorphous solid was generated, when a high percentage of sodium cholate was co-spray-dried. However, against the initial hypothesis no advantage in terms of better process efficiency was found for this sample. Amorphous solid dispersions of resveratrol and HPMC have been produced by using rotary evaporator. Conducting the HPH process with this starting material resulted in comparable mean particle sizes as compared to those obtained with unmodified starting material. Therefore, only
ACCEPTED MANUSCRIPT reduced crystallinity by using polymer also showed no beneficial effect on the particle size reduction effectiveness in this case. A comparison of the particle morphology of modified powders produced by the three methods revealed an altered and improved morphology only for the spray-dried API. This leads to the conclusion that a modified particle morphology, i.e. spherical morphology with more breakage points and
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predominantly crystal solid state (slightly reduced crystallinity but not amorphous), are the best explanation for the optimal particle size reduction effectiveness when
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spray-dried material is used. The solid states of nanosuspensions were also investigated. The results firstly confirmed that a removal of the surfactant and/or
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stabilizer from nanosuspensions prior to conducting the DSC measurement is essential for obtaining correct results. A solubility of the API in the surfactant and/or stabilizer
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might cause post-process solid state changes, which can be misleading during the
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solid state analysis of nanosuspension samples. Reference
[1] K.V. Mahesh, S.K. Singh, M. Gulati, A comparative study of top-down and bottom-up approaches for the preparation of nanosuspensions of glipizide, Powder Technol, 256 (2014) 436-449.
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[2] J. Salazar, R.H. Muller, J.P. Moschwitzer, Application of the combinative particle size reduction
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technology H 42 to produce fast dissolving glibenclamide tablets, Eur J Pharm Sci, 49 (2013) 565-577. [3] J. Salazar, O. Heinzerling, R.H. Müller, J.P. Möschwitzer, Process optimization of a novel production method for nanosuspensions using design of experiments (DoE), Int J Pharm, 420 (2011) 395-403.
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[4] T. Liu, R.H. Muller, J.P. Moschwitzer, Effect of drug physico-chemical properties on the efficiency of top-down process and characterization of nanosuspension, Expert opinion on drug delivery, 12 (2015) 1741-1754.
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[5] T. Liu, R.H. Muller, J.P. Moschwitzer, Systematical investigation of a combinative particle size reduction technology for production of resveratrol nanosuspensions, AAPS PharmSciTech, 18 (2017) 1683-1691.
[6] A. Amri, J.C. Chaumeil, S. Sfar, C. Charrueau, Administration of resveratrol: What formulation solutions to bioavailability limitations?, Journal of controlled release : official journal of the Controlled Release Society, 158 (2012) 182-193. [7] S.N. Shrotriya, N.S. Ranpise, B.V. Vidhate, Skin targeting of resveratrol utilizing solid lipid nanoparticle-engrossed gel for chemically induced irritant contact dermatitis, Drug delivery and translational research, 7 (2017) 37-52. [8] M. Milne, W. Liebenberg, M. Aucamp, The Stabilization of Amorphous Zopiclone in an Amorphous Solid Dispersion, AAPS PharmSciTech, 16 (2015) 1190-1202. [9] J.F. Willart, M. Descamps, Solid State Amorphization of Pharmaceuticals, Mol Pharmaceut, 5 (2008) 905-920.
ACCEPTED MANUSCRIPT [10] A. Singh, G. Van den Mooter, Spray drying formulation of amorphous solid dispersions, Advanced drug delivery reviews, 100 (2016) 27-50. [11] A. Sosnik, K.P. Seremeta, Advantages and challenges of the spray-drying technology for the production of pure drug particles and drug-loaded polymeric carriers, Advances in colloid and interface science, 223 (2015) 40-54. [12] S. Alaei, E. Ghasemian, A. Vatanara, Spray drying of cefixime nanosuspension to form stabilized and fast dissolving powder, Powder Technol, 288 (2016) 241-248. [13] L.A. Wegiel, L.J. Mauer, K.J. Edgar, L.S. Taylor, Crystallization of amorphous solid dispersions of resveratrol during preparation and storage-Impact of different polymers, J Pharm Sci, 102 (2013)
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[14] M. Savolainen, A. Heinz, C. Strachan, J. Yliruusi, T. Rades, N. Sandler, Screening for differences
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[15] R.C. Bennett, C. Brough, D.A. Miller, K.P. O'Donnell, J.M. Keen, J.R. Hughey, R.O. Williams, 3rd, J.W. McGinity, Preparation of amorphous solid dispersions by rotary evaporation and KinetiSol Dispersing: approaches to enhance solubility of a poorly water-soluble gum extract, Drug development
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[16] Z.N. Mahmoudi, S.B. Upadhye, D. Ferrizzi, A.R. Rajabi-Siahboomi, In vitro characterization of a novel polymeric system for preparation of amorphous solid drug dispersions, The AAPS journal, 16
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[17] A. Jahangiri, M. Barzegar-Jalali, A. Garjani, Y. Javadzadeh, H. Hamishehkar, A. Afroozian, K. Adibkia, Pharmacological and histological examination of atorvastatin-PVP K30 solid dispersions, Powder Technol, 286 (2015) 538-545.
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[18] J.E. Patterson, M.B. James, A.H. Forster, R.W. Lancaster, J.M. Butler, T. Rades, The influence of thermal and mechanical preparative techniques on the amorphous state of four poorly soluble
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compounds, J Pharm Sci-Us, 94 (2005) 1998-2012. [19] F. Lai, C. Sinico, G. Ennas, F. Marongiu, G. Marongiu, A.M. Fadda, Diclofenac nanosuspensions: influence of preparation procedure and crystal form on drug dissolution behaviour, Int J Pharm, 373 (2009) 124-132.
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[20] P. Sharma, W.A. Denny, S. Garg, Effect of wet milling process on the solid state of indomethacin and simvastatin, Int J Pharm, 380 (2009) 40-48. [21] P. Kayaert, G. Van den Mooter, Is the amorphous fraction of a dried nanosuspension caused by 650-656.
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milling or by drying? A case study with Naproxen and Cinnarizine, Eur J Pharm Biopharm, 81 (2012) [22] S. Kobierski, K. Ofori-Kwakye, R.H. Müller, C.M. Keck, Resveratrol nanosuspensions for dermal application--production, characterization, and physical stability, Pharmazie, 64 (2009) 741-747. [23] R.J. Chokshi, H.K. Sandhu, R.M. Iyer, N.H. Shah, A.W. Malick, H. Zia, Characterization of physico-mechanical properties of indomethacin and polymers to assess their suitability for hot-melt extrusion processs as a means to manufacture solid dispersion/solution, J Pharm Sci-Us, 94 (2005) 2463-2474. [24] A.B.D. Nandiyanto, K. Okuyama, Progress in developing spray-drying methods for the production of controlled morphology particles: From the nanometer to submicrometer size ranges, Adv Powder Technol, 22 (2011) 1-19. [25] M. George, I. Ghosh, Identifying the correlation between drug/stabilizer properties and critical
ACCEPTED MANUSCRIPT quality attributes (CQAs) of nanosuspension formulation prepared by wet media milling technology, Eur J Pharm Sci, 48 (2013) 142-152. [26] I. Colombo, G. Grassi, M. Grassi, Drug Mechanochemical Activation, J Pharm Sci-Us, 98 (2009)
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ACCEPTED MANUSCRIPT Highlights
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Three different methods modified resveratrol before nanocrystal production. Only reduced crystallinity showed no beneficial effect on the nanosizing efficiency. Improved morphology for spray-dried powder contributed to nanocrystal preparation. Removal of the stabilizer from nanosuspensions prior to DSC is recommended.
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Graphics Abstract
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Tao Liu, Rainer H. Müller, Jan P. Möschwitzer PII: DOI: Reference:
S0032-5910(18)30220-1 doi:10.1016/j.powtec.2018.03.028 PTEC 13259
To appear in:
Powder Technology
Received date: Revised date: Accepted date:
6 July 2017 18 February 2018 15 March 2018
Please cite this article as: Tao Liu, Rainer H. Müller, Jan P. Möschwitzer , Consideration of the solid state for resveratrol nanocrystal production. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ptec(2017), doi:10.1016/j.powtec.2018.03.028
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ACCEPTED MANUSCRIPT Consideration of the solid state for resveratrol nanocrystal production
Tao Liu 1; Rainer H. Müller 2; Jan P. Möschwitzer 2,3*
Dept. of Pharmaceutical Engineering, College of Chemical Engineering, Qingdao University of Science and
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1
Technology, Qingdao, China
Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin,
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2
Berlin, Germany
Advance Pharma GmbH and Steiner Deutsche Arzneimittel GmbH, Berlin, Germany
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*Corresponding author: Dr. Jan Peter Möschwitzer
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Tel.: +49-177-2306843
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E-mail address: [email protected]
ACCEPTED MANUSCRIPT Abstract
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Production of nanosuspension for drug bioavailability improvement has been accepted and frequently used in pharmaceutical industry. Normally, the preparation started from crystalline drug. However, some previous studies found that amorphous drug powder obtained from processes such as spray-drying might contribute to nanosizing process, i.e. reduced homogenization cycles and smaller achievable particle size. The mechanism behind this improved behavior was still unclear. In order to systematically investigate, three technologies, i.e. spray-drying, rotary evaporation and quench cooling, were used to modify drug powders, which were all processed by high pressure homogenization (HPH). Predominately amorphous solids have been obtained after processing both spray-drying (spherical particle) and rotary evaporation (no obvious morphology modification). However, those samples could not be beneficial to efficiency of nanosizing process. The smallest particle size of nanosuspensions was achieved by using spray-dried solid possessing predominately crystallinity (slightly reduced crystallinity) and changed morphology (more breakage points). Therefore, both the modification of morphology and solid state control were confirmed crucial for the followed HPH efficiency. In another part of this study, it was found that the directly dried nanosuspensions used in many previous studies were actually not suitable for a proper solid state analysis by differential scanning calorimetry (DSC). A separation step of drug and stabilizer/surfactant is strongly recommended in order to improve the validity of solid state measurement for drug nanosuspensions.
ACCEPTED MANUSCRIPT Fig. 1 Schematic description of processes for investigation.
Fig.2 DSC curves (top) and PXRD patterns (bottom) of spray-dried samples with different resveratol (RVT)/sodium cholate (SC) ratios and unmodified RVT. Fig. 3 DSC curves (top) and PXRD patterns (bottom) of rotary evaporation samples different
resveratrol
(RVT)/hypromellose
(HPMC)
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with
and
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unmodified RVT.
ratios
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Fig. 4 DSC curves (top) and PXRD patterns (bottom) of quench cooled resveratrol (RVT) and unmodified RVT.
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Fig. 5 SEM photos of unmodified resveratrol, spray-dried powders, rotary evaporation samples and quench cooled resveratrol (scale bar: 5 μm, magnification: 5000X). Resveratrol/sodium cholate ratios for spray-drying were 1:0 (A), 1:0.146 (B) and 1:0.854 (C). Resveratrol/HPMC ratios for rotary evaporation were 2:1 (D), 1:1 (E) and 1:2 (F).
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Fig. 6 Particle sizes (LD and PCS) of nanosuspensions produced from spray-dried resveratrol (RVT) with different sodium cholate (SC) ratios as well as unmodified RVT after 20 HPH cycles.
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Fig. 7 Particle sizes (LD and PCS) of nanosuspensions produced by using rotary evaporation resveratrol (RVT) with different HPMC ratios as well as
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unmodified RVT after 20 HPH cycles.
Fig. 8 Particle sizes (LD and PCS) of nanosuspensions produced by using quench cooled resveratrol (RVT) as well as unmodified RVT after 20 HPH cycles.
Fig. 9 DSC thermographs (top) and PXRD patterns (bottom) of unmodified resveratrol (A), physical mixture of resveratrol/sodium cholate in 1:1.1 (B), nanosuspension produced from unmodified resveratrol with sodium cholate (C) and sodium cholate was removed from nanosuspension (D).
ACCEPTED MANUSCRIPT 1 Introduction Bead milling and high pressure homogenization (HPH) are currently the two primary technologies for production of drug nanocrystals [1]. In addition, several combinative technologies, e.g. spray-drying [2] or freeze-drying [3] followed by HPH, have been developed. These combinative methods normally use a pre-treatment step such as
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spray-drying to modify the physical properties of drug powders prior to the top-down process. It was found that the pre-treated powders could significantly improve the
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nanosizing performance due to the reduced HPH cycles and much smaller achievable
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particle size compared to standard HPH technology [4]. For example, smaller glibenclamide nanocrystals (z-average, 236 nm) were produced by using the
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spray-dried powder compared to standard HPH (z-average, 772 nm) [2].
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A previous study showed that spray-dried drug with very low crystallinity was proven to be more effective for particle size reduction than its crystalline form [2]. This phenomenon led to the assumption that amorphous active pharmaceutical ingredient
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(API) might be beneficial for obtaining a smaller particle size and thus could improve
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the millability of the pre-treated API. However, in case of resveratrol nanocrystal production, it was difficult to build a correlation between the starting crystallinity and
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minimal achievable particle size after HPH [5]. In addition, all above related studies were only achieved by one process, i.e. spray-drying, however, amorphization can be
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achieved by various approaches based on different mechanism and consequently the obtained sample might possess different millability. Therefore, these conflicting results led to the necessity to understand the mechanism behind the improved performance systematically by comparing different pre-treatment technologies. In this regard the effect of amorphization is a key parameter to be investigated. Resveratrol is a poorly soluble plant compound, which possesses potential pharmaceutical effects. Its low bioavailability could be mainly attributed to the poor solubility and rapid metabolism [6]. Nanosizing has been proven to be an effective way to process it for medical application, such as dermal targeting of irritant contact dermatitis [7].
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Solid state modification of one compound by different methods was confirmed possible [8]. There are three rapid modification methods available, i.e. spray-drying, rotary evaporation and quench cooling, which are frequently used to modify drug
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physical properties, especially, for drug amorphization [9, 10]. These three methods constitute totally different procedures to generate amorphous APIs. Therefore,
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theoretically different kinds of modified APIs can be produced by applying those
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processes.
Spray-drying is a rapid drying technology widely used by the pharmaceutical industry.
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Drug properties e.g. particle morphology, particle size and solid state can be modified after the process [11, 12]. Spray-drying is an established technology for producing
drug
solubility
improvement.
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amorphous solid dispersions, which is a frequently used formulation technique for Various
polymers,
such
as
hydroxypropyl
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methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) have been used recently for
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the production of solid solutions [13]. In addition, carriers are not always essential for the formation of amorphous APIs. The rapid organic solvent evaporation may directly result in a modification of the solid state when spray-drying was applied [14]. As
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mentioned above, spray-drying has already been used successfully as the first step of
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the combinative particle size reduction method.
Flash evaporation by using a rotary evaporation is another way to achieve fast drying. The evaporation is achieved in a vacuum system, in which the boiling point of solvent is reduced and the corresponding drying process can be accelerated. This method can be also used to produce amorphous solid dispersion when proper drug to polymers ratios are used [15-17].
Quench cooling of a melt is another simple way for producing amorphous APIs. The molten drug is quench cooled by using liquid nitrogen. Irregularly arranged molecules
ACCEPTED MANUSCRIPT can be solidified when the cooling process is performed fast enough [14]. The process may also lead to chemical degradation because of the elevated temperatures applied [18]. However, the milling properties of those quench cooled samples are unknown as no corresponding literature has been found to date.
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In order to assess the impact of solid state changes on the particle size reduction efficiency, a proper solid state analysis is crucial for both, the unmodified and
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modified starting material, as well as for the drug nanocrystals obtained after the process. The latter characterization is important, as the solid state of drug
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nanoparticles can significantly affect the stability and dissolution rate of the nanosuspension [19]. A high energy process, such as milling, may induce a modified
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solid state of the processed material [9]. In another study, Sharma et al. have investigated the influences of HPH on the drug solid state after the process. No
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amorphous API or only a slight surface amorphization was found after the HPH
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process [20].
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To date, directly dried nanosuspensions were used in most publications to analyze the solid state of the drug nanocrystals. However, the drying process may have an influence on the solid state of API and lead to alteration as discussed above. In
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addition, the API might have solubility in the stabilizer/surfactants used for HPH. Therefore, it would be difficult to judge whether the amorphous fraction resulted from
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the milling or the downstream process [21].
The aim of this study was to investigate the resveratrol solid state throughout the nanosizing process. Spray-drying, flash evaporation by rotary evaporation and quench cooling were respectively used to modify API physical properties, i.e. solid state as well as particle morphology. All the modified powders were subsequently processed by HPH (Fig. 1). The particle sizes of nanosuspensions produced from those different starting solids were compared. In another part of this study, solid state of drug nanosuspension was carefully investigated. Two different measurement methods (with
ACCEPTED MANUSCRIPT and without stabilizer) were performed. Finally, an accurate measurement method of nanosuspension was expected.
2 Materials and methods
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2.1 Materials
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Resveratrol was obtained from E. Denk Feinchemie GmbH, Germany. Hydroxypropyl methylcellulose (HPMC) was purchased from Colorcon, England and sodium cholate
SC
from Sigma-Aldrich Chemie GmbH, Germany. Purified water was supplied from a Milli-Q system (Millipore GmbH, Germany). Cellulose nitrate membrane filters (0.1
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2.2 Resveratrol modification techniques
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μm, Ø 25mm) were purchased from Whatman GmbH, Germany.
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2.2.1 Spray-drying
Spray-drying of organic resveratrol solutions was performed by using a mini spray-dryer B-290 equipped with an inert loop B-295 (Büchi Labortechnik AG,
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Switzerland). Ethanolic solutions of resveratrol and sodium cholate in the ratios of 1:0, 1:0.146 and 1:0.854 were prepared. The following parameters of spray-drying were
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used throughout the study: inlet temperature 105oC, aspirator 80%, solution feed rate 10% and air flow 414 L/hour.
2.2.2 Rotary evaporation
Resveratrol and HPMC (2:1, 1:1 and 1:2) were dissolved in a mixture of water and ethanol (1:3). The obtained solutions were dried rapidly by a Büchi Rotavapor-R (Büchi Labortechnik AG, Switzerland) under a pressure of 100 mbar. The water bath temperature was set at 70oC and the rotary speed was set to 9.
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2.2.3 Quench cooling
Resveratrol was heated on a metal plate until the material was completely molten. Subsequently, the molten mass was immediately poured into liquid nitrogen. The
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quenched sample was collected and grinded manually in a mortar to produce
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micronized drug.
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2.3 High pressure homogenization (HPH)
A Micron Lab 40 homogenizer (APV Deutschland GmbH, Germany) equipped with a
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temperature control unit was used to produce resveratrol nanosuspensions. Firstly, the different spray-dried powders were dispersed in an aqueous sodium cholate solution
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with corresponding concentration to maintain a constant resveratrol/sodium cholate ratio of 1:1.1. Samples processed by rotary evaporation were suspended in different
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HPMC solutions to obtain suspensions with a ratio of 1:2 (resveratrol/HPMC). The
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milled quench cooled powder was suspended in an aqueous dispersion medium containing 1.1% w/w sodium cholate. The drug content of all suspensions to be nanosized by HPH was kept constant at 1.0% w/w. All coarse suspensions were first
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stirred for 1 minute by using an Ultra-Turrax T25 (IKA-Werke GmbH & Co. KG, Germany) at 8000 rpm. Subsequently, the obtained microsuspensions were
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pre-homogenized applying 2 cycles at 500 bar and 1 cycle at 1000 bar. 20 homogenization cycles at 1500 bar were employed to produce nanosuspensions at 5oC.
2.4 Differential scanning calorimetry (DSC)
The solid state of the unmodified resveratrol and all modified samples was analyzed by using a Mettler DSC 821e (Mettler Toledo, Germany). In addition, the solid state of resveratrol nanosuspension was also analyzed. The nanosuspension was dried for
ACCEPTED MANUSCRIPT measurement. Here, the resveratrol nanosuspension was handled in two different ways. One approach was to directly dry the nanosuspension to obtain powders which were subsequently analyzed by DSC. In another approach, the nanosuspension was filtered (0.1 μm) and the residual on filter membrane was washed by water in order to remove an excess of stabilizer. The drug nanoparticles without stabilizer were then collected as residuals on the membrane and then were dried. Finally, all dried samples were
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analyzed by DSC. A heating rate of 10oC/min and an accurately weighted sample
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amount of about 1-2 mg was used for all DSC measurements.
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2.5 Powder X-ray diffraction (PXRD)
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The solid state of unmodified resveratrol, modified powders and resveratrol nanosuspension were analyzed by PXRD. Measurement of nanosuspension in liquid
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state by adding viscosity enhancer (locust bean gum) in this study was found below the detection limit because of the low API concentration (no API characteristic peak
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in the pattern). Therefore, dried nanosuspension was also used for measurement. Both
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directly dried nanosuspension and stabilizer removed nanosuspension (i.e. filtered nanosuspension) were used for analysis (same sample preparation as mentioned in DSC measurement). The analysis was performed by using a Philips PW 1710
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diffractometer (Philips Industrial & Electro-Acoustic Systems Division, The
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Netherlands). The scanning angle was from 0.6º to 40º with steps of 0.02º per second.
2.6 Particle size analysis
2.6.1 Photon correlation spectroscopy (PCS)
The mean particle size (z-average) and the polydispersity index (PdI) of nanosuspensions were measured by PCS using a Zetasizer Nano ZS (Malvern Instruments, UK). Each nanosuspension of 50 µl was diluted in 3 ml purified water for 10 measurement runs in triplicate. The measurement temperature was set the same
ACCEPTED MANUSCRIPT as the sample temperature.
2.6.2 Laser diffractometry (LD)
A Mastersizer 2000 (Malvern Instruments, UK) was used to analyze the particle size
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distribution of all nanosuspensions after HPH. A real refractive index of 1.69 and an imaginary refractive index of 0.02 were used for the data calculation [22]. 3 runs were
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performed for each measurement. The obtained particle size is volume-based. LD diameter d (0.5) or d (0.9) means that 50% or 90% of the particles are smaller than the
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2.7 Scanning electron microscopy (SEM)
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given size.
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SEM was used in order to assess the particle morphology, i.e. the crystal habit of all starting materials, i.e. unmodified resveratrol, spray-dried resveratrol, rotary
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evaporation samples and quench cooled powder. A scanning electron microscope
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(Hitachi S-520, Japan) was used for the assessment. The powder was placed on a carbon holder and gold was coated with a layer thickness of 22 nm-25 nm prior to the
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analysis.
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2.8 Statistic Analysis
The statistical significance of differences between experimental groups was calculated by using Student’s t-test and p values <0.05 were considered significant.
3 Results and discussion 3.1 Effect of modification step on solid state of resveratrol As shown in Fig. 2, the unmodified resveratrol was crystalline according to both DSC and PXRD results. The corresponding DSC curve and PXRD pattern of spray-dried
ACCEPTED MANUSCRIPT resveratrol without sodium cholate were comparable to these of raw resveratrol. The spray-drying process could not alter the crystallinity of resveratrol, which means the dissolved API in ethanol tended to recrystallize during the spray-drying evaporation processes. A position shift of the melting peak was found in the DSC curve of the spray-dried sample with 1:0.146 of resveratrol/sodium cholate. However, this result
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was not matching with the PXRD patterns of the same sample, in which characteristic peaks of resveratrol could still be observed. It is assumed that resveratrol has some
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solubility in sodium cholate when the temperature was elevated during the DSC measurements. In addition, the melting point of resveratrol was decreased by sodium
SC
cholate during the heating period of DSC. A similar phenomenon was observed in other studies [20, 23]. A reduced crystallinity was found when a high amount of
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sodium cholate was used for the co-spray-drying. The DSC curve of spray-dried sample containing 1:0.854 of resveratrol/sodium cholate showed an exothermic glass
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transition peak and largely reduced melting peak. This indicated that predominately amorphous solid was achieved. The amorphous halo found in PXRD pattern of this
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sample also confirmed the DSC results. Therefore, only the spray-drying process
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could not change the crystallinity and it was essential to apply sufficient sodium cholate.
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The rotary evaporation process using HPMC as stabilizing polymer yielded solid dispersions as described in the literature[13]. It could be clearly seen from the DSC
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curves that the melting peak of resveratrol decreased as the HPMC content increased. No endothermic peak was observed in the DSC curve, when a 1:2 ratio of resveratrol/HPMC was processed (Fig. 3). This means that resveratrol molecular irregularly dispersed in the HPMC carrier and a fully amorphous solid dispersion was produced. In addition, all PXRD results were in agreement with the DSC measurements. These results confirmed that the drug to HPMC ratio had a significant influence on the solid state modification of resveratrol.
Quench cooling was also performed in order to modify the solid state of resveratrol.
ACCEPTED MANUSCRIPT Surprisingly, no reduced crystallinity was found by using this method as shown in Fig. 4. Both DSC and PXRD results of the quench cooled resveratrol were similar to these of the unmodified resveratrol.
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3.2 Particle morphology characterization of different modified powders
Fig. 5 shows the particle morphology of unmodified resveratrol and modified
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resveratrol powders yielded by the different modification processes. The spray-dried
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powders clearly showed different particle morphologies as compared to the unmodified resveratrol. A spherical structure was found for all three spray-dried
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samples. This means the droplets dissolved API during spray-drying possessed a spherical shape, which is normally the most stable shape for a droplet [24]. In addition,
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the spray-dried powders showed obvious a decrease of crystallite size. The rotary evaporation samples have also shown different particle morphologies compared to
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both the unmodified resveratrol as well as the spray-dried resveratrol. Although the
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morphology of resveratrol was totally modified after processing with a rotary evaporator, a very fragile structure could not be identified when assessing the SEM
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pictures.
The particle morphology of quench cooled sample is also shown in Fig. 5. Particle
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morphology of the quench cooled resveratrol was still similar to that of raw resveratrol with a compacted structure. Therefore, quench cooling was not a suitable process to modify the resveratrol particle morphology. 3.3 Particle sizes of nanosuspensions Comparing to the only spray-drying modification in the previous study [5], spray-drying and rotary evaporator processes produced different crystallinity of resveratrol (including the crystal sample from spray-drying without sodium cholate) by using different types (ionic and polymeric) and amounts of corresponding
ACCEPTED MANUSCRIPT excipients. In addition, both rotary evaporator and quench cooling led to different solid state as well as particle morphologies compared to the spray-dried powders and samples in previous study [5]. All the obtained samples were produced by HPH process. Fig. 6 shows the particle sizes of nanosuspensions obtained from spray-dried
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resveratrol and unmodified resveratrol after 20 homogenization cycles. The particle sizes of the predominately amorphous sample (1:0.854 resveratrol/sodium cholate)
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were 0.302 μm (d (0.5)), 1.665 μm (d (0.9)), 642 nm (z-average) and 0.306 (PdI) after
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20 homogenization cycles. These particle sizes were comparable to those yielded by the unmodified resveratrol. In contrast, the smallest particle size (d (0.5), 0.242 μm; d
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(0.9), 0.801 μm; Z-average, 356 nm; PdI, 0.113) was achieved by using predominately crystalline spray-dried sample (1:0.146) (p<0.05). The particle size of spray-dried
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resveratrol without sodium cholate was also smaller compared to the result of a nanosuspension generated with unmodified resveratrol after 20 homogenization
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cycles (p<0.05), although the solid state was similar to unmodified resveratrol.
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Therefore, it confirmed the necessity of morphological improvement for an effective particle size reduction during HPH process, which could not be clearly answered in the former related study [5].
It could be possible that the improved performance of
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this spray-dried sample might result from the increase of crystal weak points or decrease of crystallite size as shown in SEM photos. Although those three spray-dried
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powders showed comparable morphology, the one with lowest crystallinity could not be beneficial for achieving an improved particle size reduction effectiveness and one possible reason was that the recrystallization happened during the HPH process.
Fig. 7 shows the particle sizes of resveratrol nanosuspensions from the rotary evaporation samples compared to unmodified resveratrol. The obtained mean particle sizes (z-average) after 20 homogenization cycles by using solid dispersion of resveratrol/HPMC (1:2, 1:1 and 2:1) were 559 nm (PdI, 0.148), 558 nm (PdI, 0.152), and 520 nm (PdI, 0.136), respectively. These results were comparable to the particle
ACCEPTED MANUSCRIPT size of unmodified resveratrol nanosuspension (579 nm) (p>0.05). The LD results also confirmed that all particle sizes of modified samples were similar to the unmodified one. As shown in Fig. 7 in general the LD d (0.9) value of rotary evaporation samples were smaller than that of the unmodified resveratrol, but d (0.5) values became larger. The generation of amorphous solid dispersions by using
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additional polymer (HPMC) could not contribute to the obtained average nanoparticle size according to both PCS and LD. Those results from rotary evaporation samples
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showed that the amorphization method and added excipient could be also crucial for drug modification. Although crystallinity at different levels was achieved by rotary
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evaporation with polymer (HPMC), it could not influence the minimal achievable
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particle size positively.
The particle size of quench cooled resveratrol after 20 homogenization cycles was
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comparable to that of unmodified resveratrol nanosuspension according to both LD and PCS measurements (p>0.05) (Fig. 8). When comparing this sample and the
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spray-dried sample without sodium cholate, the solid state of both were similar,
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however, the particle morphologies were found to be totally different. In addition, some recent studies found that the particle morphology of starting drug powder was an important parameter of the nanocrystals production [3, 25]. Therefore, it can be
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concluded that the modification of the resveratrol morphology played an important
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role for the subsequent HPH process.
According to the process performance when processing these three kinds of modified powders, only spray-dried samples contributed to an improved process efficiency compared to unmodified resveratrol. The modification of solid state by using rotary evaporation could not be directly correlated with the final nanoparticle size. Therefore, it is also proved that a valid modification of resveratrol for the followed HPH was highly dependent on the amorphization method and applied co-processed excipient. The modified morphology of spay-dried powder was also an important aspect for achieving a smaller particle size. In order to obtain the smallest particle in
ACCEPTED MANUSCRIPT nanosuspension, the powder used for HPH was expected to possess modified morphology (spray-dried, more breakage points) as well as predominantly crystal solid state (slightly reduced crystallinity but not amorphous).
3.4 Characterization of resveratrol solid state in nanosuspension
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Conflicting data have been found with regard to the solid state of the resveratrol nanocrytals after the HPH process. An exothermic peak and a very small melting peak
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were found in DSC thermogram (C in Fig. 9) of unmodified resveratrol
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nanosuspension. This would normally indicate a solid state change towards a predominately amorphous solid. However, the characteristic peaks in PXRD
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diffractograms (C in Fig. 9) indicated a predominately crystalline state of this particular sample. As the directly dried nanosuspension contained resveratrol and
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sodium cholate simultaneously, it was hypothesized that the presence of sodium cholate could have led to incorrect DSC results.
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In order to test this hypothesis, sodium cholate was removed to a great extend by
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filtration to exclude any potential risk of an alteration of the solid state of resveratrol during the DSC heating. The results for this study are shown in Fig. 9 (D). The filtrated sample only showed one melting peak and a slight reduction of melting point
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(266oC) which might be attributed to the smaller particle size [26]. The DSC and
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PXRD results were in good agreement when the filtrated samples have been analyzed. Additionally, a physical mixture of resveratrol and sodium cholate (1:1.1) was investigated (B in Fig. 9). The PXRD pattern of the nanosuspension was comparable to the physical mixture of resveratrol and sodium cholate. Two melting peaks (108oC and 243oC) were found in the DSC thermograph. The melting point of resveratrol (243oC) was lower than that of unmodified resveratrol. This reduction of melting point has also confirmed the solubility of resveratrol in sodium cholate during the DSC heating period. The lower melting temperature of the dried nanosuspenison compared to the physical mixture might be again a result of the smaller particle size
ACCEPTED MANUSCRIPT of resveratrol. Here it can be assumed that the solubility of resveratrol in the stabilizer, i.e. sodium cholate, was further improved due to the smaller particle size of resveratrol.
The phenomenon shown above confirmed that, if the nanosuspension cannot be
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measured in liquid state due to the low API concentration, the directly dried nanosuspensions used in most previous studies were actually not suitable for a proper
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solid state analysis by DSC. If the API has the solubility in the specific surfactant or polymer used for the physical stabilization of the particles, a kind of solid dispersion
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can be generated during the heating period of the DSC measurement. The real solid state of an API could be masked by using this analytical procedure. This can result in
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misleading conclusions. This problem about DSC measurement of nanosuspension was firstly proposed in this study, which has not been aware in most related
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nanocrystal publications. Therefore, it is strongly recommended to remove an excess of surfactant, stabilizer and polymer from the nanosuspension by filtration or other
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purification methods before DSC measurement.
4 Conclusion
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The resveratrol powders modified by spray-drying, rotary evaporation and quench cooling were nanosized by HPH, respectively. Spray-dried powders containing tiny
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amounts of sodium cholate or without sodium cholate showed predominately crystalline solid state. Using these samples, the HPH process was more efficient compared to all other modified or unmodified starting materials. Predominately amorphous solid was generated, when a high percentage of sodium cholate was co-spray-dried. However, against the initial hypothesis no advantage in terms of better process efficiency was found for this sample. Amorphous solid dispersions of resveratrol and HPMC have been produced by using rotary evaporator. Conducting the HPH process with this starting material resulted in comparable mean particle sizes as compared to those obtained with unmodified starting material. Therefore, only
ACCEPTED MANUSCRIPT reduced crystallinity by using polymer also showed no beneficial effect on the particle size reduction effectiveness in this case. A comparison of the particle morphology of modified powders produced by the three methods revealed an altered and improved morphology only for the spray-dried API. This leads to the conclusion that a modified particle morphology, i.e. spherical morphology with more breakage points and
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predominantly crystal solid state (slightly reduced crystallinity but not amorphous), are the best explanation for the optimal particle size reduction effectiveness when
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spray-dried material is used. The solid states of nanosuspensions were also investigated. The results firstly confirmed that a removal of the surfactant and/or
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stabilizer from nanosuspensions prior to conducting the DSC measurement is essential for obtaining correct results. A solubility of the API in the surfactant and/or stabilizer
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might cause post-process solid state changes, which can be misleading during the
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solid state analysis of nanosuspension samples. Reference
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ACCEPTED MANUSCRIPT Highlights
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Three different methods modified resveratrol before nanocrystal production. Only reduced crystallinity showed no beneficial effect on the nanosizing efficiency. Improved morphology for spray-dried powder contributed to nanocrystal preparation. Removal of the stabilizer from nanosuspensions prior to DSC is recommended.
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Graphics Abstract
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