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Flavonoid nanocrystals produced by ARTcrystal®-technology
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IJP 14447 1–11 International Journal of Pharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Pharmaceutical nanotechnology
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Flavonoid nanocrystals produced by ARTcrystal1-technology
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Patrik Scholz a,b , Cornelia M. Keck a,b, *
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a b
University of Applied Sciences Kaiserslautern, Applied Pharmacy Division, Campus Pirmasens, Germany Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 September 2014 Received in revised form 2 November 2014 Accepted 4 November 2014 Available online xxx
ARTcrystal1-technology is a novel technique for a more efficient production of nanocrystals. It consists of a high speed stirring (HSS) step as pre-milling and subsequent high pressure homogenization (HPH) at reduced pressure and cycle numbers. In this study, three antioxidants, rutin, hesperidin and apigenin, were processed by ARTcrystal1-technology, the results were compared to sizes obtained for the production of nanocrystals produced by classical HPH. By using the ARTcrystal1-process, all three substances could be transformed into nanosuspensions with mean sizes and PdIs of 431 nm/0.27, 717 nm/ 0.21 and 262 nm/0.31, respectively. Depending on the properties of the raw material the ARTcrystal1technology revealed similar or even better results than classical HPH. Further optimization of the setup of the HSS process might lead to an optimized process with higher efficacy than classical HPH. ã 2014 Published by Elsevier B.V.
Keywords: ARTcrystal Nanocrystal Rutin Hesperidin Apigenin
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1. Introduction
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Flavonoids, e.g., rutin, hesperidin and apigenin possess many positive biological effects. They scavenge reactive oxygen species, such as hydroxyl radicals and superoxide radicals, thus preventing biological and chemical substances from oxidative damage. These radicals can come into existence by UV-radiation. Furthermore, rutin has antiplatelet, antiviral and antihypertensive effects (Ghiasi and Heravi, 2011). Flavonoids have a light tumor growth inhibition potential (Deschner et al., 1991). Due to their manifold benefits, flavonoids have a great potential for supporting various disease treatments, thus being interesting substances in the pharmaceutical and nutraceutical field. However, a limiting factor is their poor solubility, restricting their full antioxidative potential as well as their bioavailability. To overcome this problem the solubility of the actives needs to be increased. During the last decades different ways to enhance the solubility of poorly soluble actives were developed. Examples are soluble drug salts, co-solvents or cyclodextrins (Frömming and Frömming, 1993). However, one of the most successful methods is the production of nanocrystals. Nanocrystals consist of 100% pure drug, only being stabilized by a small amount (1–2% (w/w)) of stabilizer and possess a mean crystal size below 1000 nm, typically between 200 and 500 nm (Keck and Müller, 2006). In contrast to
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* Corresponding author at: Fachhochschule/University of Applied Sciences Kaiserslautern, Campus Pirmasens – Applied Pharmacy, Carl-Schurz-Str. 10–16, 66953 Pirmasens, Germany. Tel.: +49 631 3724 7031; fax: +49 631 3724 7044. E-mail address: [email protected] (C.M. Keck).
larger crystals, nanocrystals possess a higher curvature. According to the Ostwald–Freundlich equation, this leads to a higher dissolution pressure and thus to a higher kinetic saturation solubility (Wu and Nancollas, 1998). Furthermore, nanocrystals possess a larger surface area. Thus, according to the Noyes–Whitney equation this leads to an increased dissolution velocity. Finally, the greater surface-to-mass ratio enables mucosal adherence of nanocrystals, allowing them to stay in the upper parts of the gastrointestinal tract until complete dissolution is ensured. As a consequence, the possibility of a fast gastrointestinal passage and fecal elimination of undissolved crystals is reduced and bioavailability is increased (Müller et al., 2011). In fact, the production of nanocrystals is the most elegant way to overcome poor solubility and to increase to bioavailability of poorly soluble actives. There are two main approaches of nanocrystal production, the bottom-up and the top-down approach. While the bottom-up process focuses on in situ forming of nanocrystals by precipitation, top-down processes produce nanocrystals by grinding large crystals into the nanoscale. Common top-down techniques are high pressure homogenization (HPH) and bead milling (Müller et al., 2011). Newer processes combine a pre-treatment step with subsequent HPH. Examples are freeze drying and HPH or bead milling and HPH (Salazar et al., 2014). These combination techniques aim at achieving smaller crystal sizes. Recently another combination process was introduced. This process is called the ARTcrystal1-technology and aims at a faster and more economic nanocrystal production compared to traditional top-down techniques. It combines an effective rotor–stator high speed stirring (HSS) process with subsequent HPH at reduced
http://dx.doi.org/10.1016/j.ijpharm.2014.11.008 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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numbers and cycles (Keck, 2011). Recent studies investigated the major process parameters of the HSS process, such as in process temperature and HSS pretreatment efficacy depending on raw material properties (Scholz et al., 2014) for the flavonoid rutin. To further prove the efficacy of the ARTcrystal1-technology, this study aimed at investigating the efficacy of the HSS process in comparison to the classical pre-milling step of HPH and to apply the method to different materials, i.e., to other flavonoids with different physicochemical properties. Finally, it was aimed at comparing the results obtained by ARTcrystal1-technology to the results obtained by classical HPH.
2. Materials and methods
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2.1. Materials
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Coarse rutin (Art.-No. 907861) and jet-milled rutin (Art.-No. 900313) were purchased from Denk Ingredients, Munich, Germany. Apigenin and hesperidin were purchased from Exquim, S.A., Barcelona, Spain. As stabilizer Plantacare1 2000 (decyl glucoside, BASF, Ludwigshafen, Germany) was used. Distilled water was obtained from a WDA 25 distillation unit (QVF PILOTTEC, Jena, Germany). All other chemicals were used as received.
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Fig. 1. DLS data (upper) and LD data (bottom) for coarse rutin processed by ARTcrystal1-technology compared to coarse rutin processed by the classical pre-milling step (rotor–stator mixing and HPH at low pressures).
Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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2.2. Methods
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In the first part of the study, the efficacy of the high speed stirring (HSS) step was compared to the efficacy of the classical pre-milling step of HPH. For this a coarse rutin suspension was pretreated by a classical high speed rotor–stator stirrer and premilled by HPH at low pressures. Results were compared to coarse rutin processed by ARTcrystal1-technology, i.e., effective rotor–stator high speed stirring and subsequent HPH at low pressures. In the second part, different flavonoids were processed by the ARTcrystal1-technology and the results were compared to results obtained by classical HPH. Finally the physical stability of the nanosuspensions was investigated to prove the absence of stability issues caused by Ostwald ripening or agglomeration.
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2.2.1. Preparation of macrosuspensions All suspensions contained 5% (w/w) drug material as well as 1% (w/w) Plantacare1 2000 as stabilizer and distilled water to up to 100.0%. Macrosuspensions were produced by dispersing the dry powder in the surfactant solution under magnetic stirring. The coarse suspensions were stored at 4 C in a refrigerator until use. 2.2.2. Production of nanosuspensions by ARTcrystal1-technology 1 liter of macrosuspension was processed in an ART MICCRA D27 rotor stator system (ART Labor- und Prozesstechnik, Müllheim, Germany). High speed stirring (HSS) as pretreatment prior to HPH was performed by using a rotor unit with a slit size of 1 mm and a stator with a slit size of 0.5 mm. The gap width between rotor and stator was 0.1 mm. Return flow to the product container was modulated by a valve position of 45 for reduction of foam formation (Scholz et al., 2014). HSS was performed for 5 min at 24,000 rpm. During the production the D27 system was cooled to 10 C using an Alpha RA12 cooling device (Lauda, LaudaKönigshofen, Germany). In-process temperature was controlled by a thermometer HI 8314 (HANNA Instruments, Kehl, Germany). After this HSS step, subsequently HPH was applied for 5 cycles at 300, 500 or 750 bar in an APV LAB 40 high pressure homogenizer (APV Deutschland GmbH, Germany). Cooling of the samples between the cycles was provided for stability reasons (Keck, 2006). 2.2.3. Production of suspensions by classical pre-milling of HPH Production, i.e., pre-milling, was performed as described in the literature (Mishra et al., 2009; Chen, 2013). Briefly, macrosuspensions were subjected to pre-mixing by using a rotor–stator dispersing unit (D9, ART Labor- und Prozesstechnik, Müllheim, Germany) at 39,000 rpm for 1 min. Subsequently, the suspension was subjected to HPH at low pressures, i.e., 2 cycles at 250 bar, followed by 5 cycles at 500 bar.
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2.2.4. Short-term physical stability Since stability issues are very important for pharmaceutical products, short term physical stability of the produced nanosuspensions was investigated for a period of 30 days when stored at room temperature.
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2.3. Particle size analysis
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Samples were analyzed regarding size, size distribution and morphology using dynamic light scattering, laser diffraction and light microscopy.
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particle size and the polydispersity index (PdI) as a measure for the width of the size distribution. Prior to the measurements, samples were diluted in saturated solutions of the given substance by a factor of 1:200. All measurements were performed at 25 C in triplicates. Results were analyzed by using the general purpose mode.
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2.3.2. Laser diffraction For laser diffraction (LD) a Mastersizer 2000 with Hydro S wet dispersion unit (Malvern Instruments, UK) was used. LD yields a volume based particle size distribution and is capable to analyze particles in the microscale as well as in the nanoscale size range. For particle population characterization, the volume-weighted diameters d(v) 0.50, d(v) 0.95 and d(v) 0.99 were chosen. All measurements were performed as triplicates in saturated drug
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Table 1 Particle size (LD data) and microscopic images (magnification 1000 fold) of the raw materials of coarse rutin, jet-milled rutin, hesperidin and apigenin. Material
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d(v) 0.50 d(v) 0.99 Microscopic image (1000 fold) (mm) (mm)
Rutin (coarse)
5.49
61.64
Rutin (jet milled)
10.50
129.16
4.96
86.73
55.90
150.23
Hesperidin
Apigenin
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2.3.1. Dynamic light scattering Dynamic light scattering (DLS) measurements were performed by a Zetasizer Nano ZS (Malvern Instruments, UK). DLS yields the intensity based hydrodynamic diameter (z-average) as mean Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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medium as dispersion medium (Keck, 2010). Mie theory was applied for size analysis by using the optical parameters (real/ imaginary refractive index) 1.593/0.02 for rutin (Keck, 2010), 1.59/ 0.01 for apigenin (Al Shaal et al., 2010) and 1.57/0.01 for hesperidin (Petersen, 2008). 2.3.3. Light microscopy Light microscopy was used to characterize particle morphology and to prove the results obtained by LD analysis (Keck and Müller, 2008; Keck, 2010). A DM1000 light microscope with MD170HD camera and Leica Application Suite software (Leica Microsystems, Wetzlar, Germany) was utilized for this purpose. 200 fold magnification was applied for overviewing the sample and detecting larger particles or agglomerates and 1000 fold
magnification was used for detailed characterization of the particles.
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3. Results and discussion
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3.1. Comparison of diminution efficacy of high speed stirring (HSS) vs. classical pre-milling
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Classical pre-milling was performed by rendering a coarse rutin suspension with a rotor–stator pre-mixing step (39,000 rpm, 1 min) and subsequent HPH (2 cycles at 250 bar, 5 cycles at 500 bar). ARTcrystal1-technology was applied by applying HSS for 5 min at 24,000 rpm, followed by 5 cycles of HPH at 500 bar. The results obtained are shown in Fig. 1.
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Fig. 2. DLS data (upper) and LD data (bottom) for coarse (left) and jet milled (right) rutin processed by ARTcrystal1-technology for up to 5 cycles at 300 bar and 500 bar.
Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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HSS was found to be more efficient than the classical premixing. Whereas the HSS led to a suspension with a d(v) 0.50 of 3.68 mm and a d(v) 0.99 of 28.6 mm (Fig. 1, left), the classical premixing, i.e., 39.000 rpm for 1 min, resulted in a d(v) 0.50 of 5.58 mm and a d(v) 0.99 of 125.85 mm (Fig. 1, right). Pre-milling for 2 cycles at 250 bar decreased the d(v) 0.50 to 3.18 mm and the d(v) 0.99 to 18.9 mm. Hence, about similar results were obtained by either applying HSS for 5 min or the classical pre-milling, e.g., 1 min stirring at 39,000 rpm and 2 cycles at 250 bar. Further processing at 500 bar HPH resulted in a further decrease in size for both suspensions. After 1 cycle at 500 bar no significant differences were obtained between the two samples. However, after 5 cycles at 500 bar samples processed with HSS were found to possess much smaller sizes and a narrower size distribution than the suspension processed with the classical pre-treatment.
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The HSS treated suspension led to a d(v) 0.50 of 0.39 mm, a z-average of 431 nm and a PdI of 0.27, whereas the suspension pretreated with the classical milling led to a d(v) 0.50 of 1.48, a z-average of 470 nm and a PdI of 0.41. Both suspensions contained larger remaining particles. However, the particles for the HSS pretreated suspension were distinctly smaller, than the particles which remained in the suspension pre-treated with the classical milling process. A possible explanation for the higher efficacy of the HSS might be that HSS was more efficient in destroying larger crystals and Q2 agglomerates. However, it might also be possible the HSS pretreatment created imperfections and damages in the crystal structures by hydrodynamic shearing. Hence, even though crystals could not be fully diminuted by HSS, they could be easily broken down by subsequent HPH. Classical pre-mixing at Q3
Table 2 Microscopic images of coarse and jet milled rutin: raw material prior to processing, after 5 min of HSS pretreatment and after 5 cycles HPH at 300 bar or 500 bar (magnification 1000). Jet milled rutin
Coarse rutin
Raw material
ARTcrystal1-pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 300 bar
ARTcrystal1 HPH 5 500 bar
Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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39,000 rpm by the D9 sowed only a small effect on particle size reduction due to turbulences inside the beaker, i.e., not every particle was forced to pass the rotor. Thus, even elongation of stirring would not result in a fine suspension. Additionally, the rotor inside the D9 is distinctly smaller, leading to less shear stress due to lower shear tip speed and thus reduced size reduction efficacy (Rodgers and Cooke, 2012). 3.2. Production of different flavonoid nanocrystals by ARTcrystal1technology To further investigate the nanonization potential of the ARTcrystal1-technology, different flavonoids with different physical properties, such as crystal size and morphology were processed. The aim was to determine the influence of different material properties, such as starting particle size and shape on
final crystal size after processing. Four flavonoid materials were selected and the raw material properties were analyzed by means of particle size and morphology. The results are shown in Table 1. As displayed in Table 1, the four raw materials differ in their properties. Coarse rutin contained mostly large monolithic, cuboid crystals with sizes of about 30 mm in length and of about 5 mm in diameter. Jet milled rutin raw material consisted mostly of very fine needles with submicron diameter and some large aggregates. Hesperidin raw material contained bundle-like shaped aggregates of thin needle-shaped crystals. Apigenin raw material consisted of very large crystals, as indicated by light microscopy and a d(v) 0.50 of 55.9 mm. Due to the different properties the flavonoids were found to be suitable to investigate if the ARTcrystal1-method is able to nanonize different raw materials with different physical properties, such as crystal size and morphology.
Fig. 3. DLS data (upper) and LD data (bottom) for hesperidin processed by ARTcrystal1-technology at 300, 500 and 750 bar.
Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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In the first study, coarse and jet milled rutin material were used to determine the influence of the size and crystal morphology of the starting material on the final product size. Results are shown in Fig. 2. 3.2.1. Coarse rutin Subjecting the coarse rutin suspension to HSS resulted in a reduction of the d(v) 0.99 from 61.6 mm to 28.6 mm after 5 min of stirring (Fig. 2, left). One cycle at 300 bar reduced the d(v) 0.99 of 21.4 mm, further processing resulted after 5 cycles in a d(v) 0.50 of 1.58 mm and a d(v) 0.99 of 9.57 mm. DLS measurements revealed a z-average of 471 nm and a PdI of 0.342, indicating the resulting suspension is in the submicron scale. However, the PdI was found to by >0.3. Thus, the suspension is very polydisperse. Increasing the pressure to 500 bar could further decrease the particle size. One cycle of HPH at 500 bar resulted in a d(v) 0.99 of 17.3 mm. Further processing led to a stronger decrease in particle size, especially in d(v) 0.99. After 5 cycles at 500 bar, the resulting nanosuspension possessed a z-average of 431 nm, a PdI of 0.272, a d (v) 0.50 of 0.39 and a d(v) 0.99 of 6.23 mm (Fig. 2, right). The resulting suspension still contained some microscale crystals, as proven by LD data and light microscopy (Fig. 2, Table 2). Nevertheless, the resulting suspension can be considered as a nanosuspension. 3.2.2. Jet milled rutin For the jet milled material, a strong reduction in size from 129.2 mm down to 4.5 mm (d(v) 0.99) within 5 min of HSS was obtained. This suspension already possessed nanoscale dimensions (572 nm, PdI of 0.29, Fig. 2, left). Subjecting this suspension after HSS pretreatment to HPH at 300 bar led to a further reduction of the d(v) 0.99 as well as to a reduction of the z-average. The resulting nanosuspension had a mean particle size of 488 nm, a PdI of 0.29 and a d(v) 0.99 of 3.37 mm. Additional cycles at 300 bar further reduced the crystal size slightly, mostly by destroying some of the remaining microcrystals. After 5 cycles at 300 bar, a nanosuspension with a z-average of 427 nm, a PdI of 0.21 and a d(v) 0.99 of 3.06 mm was obtained. The further reduction of PdI (from 0.29 after HSS to 0.21 after HPH at 300 bar) and d(v) 0.99 (from 4.54 mm to 3.06 mm) indicated an increase in homogeneity. Processing the pretreated suspension at 500 bar resulted in a small further decrease in size and a further slight increase in suspension homogeneity. After 5 cycles, a z-average of 411 nm, a PdI of 0.23 and a d(v) 0.99 of 2.84 mm could be achieved, revealing that 300 and 500 bar HPH led to nearly similar results. The differences in the results after processing coarse and jet milled rutin can be explained by light microscopy (Table 2). The jet milled material contained very large aggregates combined with a large amount of fine needles. The coarse material included nearly no aggregates, but large monolithic crystals. The fine needle aggregates can be destroyed with sufficient hydrodynamic shear forces, whereas the monolithic crystals need to be subjected to stronger forces, such as cavitation forces, in order to be broken down into crystals with desired sizes. Both, hydrodynamic shear forces and cavitation, occur in HPH (Keck and Müller, 2006), while particle breakdown in the high speed stirring is achieved mainly by hydrodynamic shear forces and minor cavitation forces (Badve et al., 2014; Jasinska et al., 2014). Thus, lager crystals require more energy input by HPH. Aiming at an increase in diminution efficacy of HSS, HSS at increased stirring rates (up to 36,000 rpm) or using rotors and stators with modified geometries and surfaces should be applied.
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nanosuspension (d(v) 0.50 1.84 mm, d(v) 0.99 7.49 mm, z-average 794 nm, PdI 0.54). Even HPH for 5 cycles at 500 bar was not sufficient to form a nanosuspension with a sufficient small size and size distribution (d(v) 0.50 of 1.49 mm, d(v) 0.99 of 5.84 mm, z-average 666 nm, PdI 0.34). To investigate if a higher energy input would lead to a smaller size, additional 5 cycles at 750 bar were
Table 3 Microscopic images of hesperidin: raw material prior to processing before processing, after 5 min of HSS pretreatment, and after 5 cycles of HPH at 300, 500 or 750 bar (magnification 1000). Raw material
ARTcrystal1 HSS pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 300 bar
ARTcrystal1 HPH 5 500 bar
ARTcrystal1 HPH 5 750 bar
3.2.3. Hesperidin After HSS the hesperidin microcrystals possessed a particle size distribution almost similar to the one of coarse rutin material (Fig. 3). The d(v) 0.50 was 3.34 mm and the d(v) 0.99 was 22.91 mm. Further processing for 5 cycles at 300 bar did not lead to a Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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applied. Indeed, the increase of the pressure to 750 bar resulted in a nanosuspension with a d(v) 0.50 of 0.94 mm, a z-average of 717 nm and a narrow PdI of 0.21. The efficient reduction in size was confirmed by light microscopy (Table 3). The raw material contained large aggregates of long rod-shaped crystals with a length of about 30 mm. After 5 cycles at 500 bar, the resulting nanosuspension contained only a small amount of microscale crystals (Table 3). In accordance to the results from the rutin nanosuspension, it can be concluded that HSS and subsequent HPH at 300 bar is not always powerful enough to form nanosuspensions from very coarse material. The coarse material contained large crystals of up to 100 mm, which could be broken down to around 22 mm after HSS. HPH at low pressures, such as 300 bar, seems not to be able to destroy these large crystals within 5 cycles. Increasing the energy input by processing 10 or 20 cycles and/or by increasing the homogenization pressure may result in nanosuspensions with smaller sizes. 3.2.4. Apigenin According to the results obtained from rutin and hesperidin, applying ARTcrystal1-technology with HPH at 300 bar pressure
did not seem suitable for raw materials consisting of mainly large crystals. Thus, for apigenin possessing a d(v) 0.50 of >50 mm (c.f. Table 1), the application of low pressure, i.e., 300 bar seemed to be not sensible, thus only pressures of 500 and 750 bar were applied. In contrast to hesperidin, after HSS for apigenin, a small d(v) 0.50 of 1.35 mm and a large d(v) 0.99 of 60.39 mm was found (Fig. 4). DLS results revealed a mean particle size of 327 nm and a PdI of 0.38. After only 1 cycle of HPH at 500 bar, the d(v) 0.50 was reduced to 0.48 mm, yet still large particles being present (d(v) 0.99 30.78 mm). After 5 cycles at 500 bar, the d(v) 0.99 was reduced to 8.43 mm with a z-average of 269 nm and a PdI of 0.33. Increasing the pressure to 750 bar, 5 cycles of HPH resulted in a nanosuspension with a d(v) 0.50 of 0.15 mm, a d(v) 0.99 of 6.21 mm, a z-average of 262 nm and a PdI of 0.318. Light microscopy shows the different material properties compared to hesperidin (Table 4). While hesperidin formed large bundle-shaped aggregates, apigenin raw material consisted of large, dense aggregates. Thus, it can be concluded that HSS and subsequent HPH are more efficient in diminuting larger crystals than agglomerates of needle like material. A possible explanation is the orientation of needles in
Fig. 4. DLS data (upper) and LD data (bottom) for apigenin processed by ARTcrystal1-technology at 500 and 750 bar.
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ARTcrystal1-pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 500 bar
ARTcrystal1 HPH 5 750 bar
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the rotor–stator flow, allowing to bypass areas of high shear intensity due to their small diameter. To counteract this, a redesign of the rotor–stator geometry, e.g., by reducing the slit size, might enable to prevent particles from passing through areas of low shear intensity, thus leading to a more efficient reduction in size during the HSS pretreatment step.
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3.3. Comparison of ARTcrystal1-technology with classical HPH
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To estimate the efficacy of the Artcrystal-technology, the results obtained from this study were compared to results obtained by classical HPH. The results are summarized in Table 5. Compared to classical HPH (pre-milling and 20 cycles at 1500 bar), the ARTcrystal1-technology was found to lead to better results. By using the ARTcrystal-technology a coarse rutin nanosuspension with a size of 431 nm, a PdI of 0.27 and a d(v) 0.99 of 6.23 mm was produced, while classical HPH achieved a size of 727 nm with a PdI of 0.27 and a d(v) 0.99 of 2.40 mm (Mauludin et al., 2010). In fact, ARTcrystal-technology produced a nanosuspension with a distinctly smaller mean size while reaching the same homogeneity. However, the ARTcrystaltechnology was less sufficient in reducing the amount of remaining larger crystals. Jet milled rutin was processed by ARTcrystal1-technology to a nanosuspension with a size of 411 nm, a PdI of 0.23 and a d(v) 0.99 of 2.84 mm. Processing jet milled rutin by classical HPH resulted in a nanosuspension with a z-average of 381 nm and a very high PdI of 0.8 and a d(v) 0.99 below 1.5 mm. Hence, ARTcrystal1-technology formed a nanosuspension with improved homogeneity in comparison to HPH, discernable by the PdI values. The high PdI from HPH indicated agglomeration, which could not be detected by LD due to the influence of stirrer in the dispersion unit (Chen, 2013). Agglomeration is often caused by too high production temperatures which occur due to the high dissipation during HPH (Keck, 2006). In contrast, ARTcrystaltechnology only applies low pressures, thus less dissipation occurs during the production. Therefore, the ARTcrystal-process might be especially suitable for materials being sensitive to agglomeration during production. Hesperidin processed by ARTcrystal1 technology at 750 bar resulted in a nanosuspension with a size of 717 nm, a PdI 0.21 and a d(v) 0.99 of 4.35 mm. Processing of hesperidin suspensions, stabilized with either Poloxamer1 188, sodium dodecyl sulfate, Tween1 80 or polyvinyl alcohol, by HPH, resulted in crystal sizes of 239–449 nm, PdIs of 0.308–0.434 and d(v) 0.99 of 2.06–5.63 mm (Mauludin and Müller, 2013). While the mean particle size is larger compared to traditional HPH, a lower PdI could be achieved by ARTcrystal1-technology. Also these data indicate that the ARTcrystal process enables the production of suspensions with a narrower size distribution, when compared to classical HPH. However, the efficacy of destroying all larger particles seems to be slightly lower in comparison the classical HPH. These results were also confirmed by the results obtained for the apigenin nanosuspension. ARTcrystal1-technology produced a nanosuspension with a mean size of 262 nm, a PdI of 0.32 and a d(v) 0.99 of 6.21 mm. Classical HPH led to sizes of 484 nm, a PdI of 0.355 and a d(v) 0.99 of 2.78 mm (unpublished data). The data obtained from this study suggest to optimize the ArtCrystal process in regard to increase the efficacy in eliminating larger remaining particles. In case of classical HPH this optimization was achieved by combining bead milling and HPH (Petersen, 2008). This process is known as smartCrystal1 combination technique. Processing apigenin via this process led to a size of
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Table 5 Comparison of particle sizes obtained after production of nanosuspensions by ARTcrystal1-technology and classical HPH (data obtained from literature). Material
ARTcrystal1-technology (z-average, PdI)
Literature (z-average, PdI)
Rutin (coarse) Rutin (jet milled) Hesperidin Apigenin
431 nm, 0.27 411 nm, 0.23 717 nm, 0.21 262 nm, 0.32
727 nm, 0.27 (Mauludin et al., 2010) 381 nm, 0.8 (Chen, 2013) 239–449 nm, PdI 0.31–0.43 (Mauludin and Müller, 2013) 439 nm, 0.28 (Al Shaal et al., 2011)
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439 nm, a PdI of 0.28 and a d(v) 0.99 of 0.52 mm (Al Shaal et al., 2011). However, the smartCrystal1 process is time consuming, as it involves the bead milling as pre-treatment step. In contrast, the ArtCrystal process is extremely fast, i.e., it takes only a few minutes. Thus, if the efficacy of diminuting larger remaining crystals can be increased, the ArtCrystal process will be superior in comparison to classical approaches for the production of nanosuspensions. Earlier studies already indicated that elongation of the HSS stirring time might destroy remaining crystals with sizes above approximately 10 mm (Scholz et al., 2014). Alternatively, an increase of stirring speed to 36,000 rpm might allow faster and better destruction of larger particles. Further optimization potential lies in the optimization of the rotor and stator geometry, e.g., the reduction in the slit size (Scholz and Keck, 2014).
3.4. Short-term physical stability of the produced rutin nanosuspensions
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Since physical stability is an important issue a short term physical stability study was conducted. The aim of this stability study was to prove that the ARTcrystal1-technology is able to produce stable nanosuspensions. Results are displayed in Fig. 5. The results showed that the nanosuspensions were stable within 30 days. However, slight increases in size were observed for all suspensions. For both rutin nanosuspensions DLS data increased, whereas no changes were detected by LD measurements. Light microscopy revealed a slight agglomeration of the particles. The agglomerates were very loose and could be easily de-aggregated by light shacking. Therefore, these agglomerates could also be destroyed by light stirring in the wet dispersion unit of the LD instrument, thus
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Fig. 5. DLS data (top) and LD data (bottom) for short term physical stability (30 days) of flavonoid nanosuspensions (hesperidin, apigenin, jet milled and coarse rutin) processed by ARTcrystal1-technology.
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LD could not detect any change in particle size. However, DLS measurements are performed without stirring. Thus, during the DLS measurements no stirring induced hydrodynamic shear stress is applied to the sample, which consequently enables the detection of agglomerates. The agglomeration was more present in the jet milled suspension. A possible explanation for this is the needle like structure of the jet milled rutin, which possesses a greater surfaceto-mass ratio than the more cuboid crystals of the coarse rutin, thus they tend to agglomerate more easily (Gao et al., 2004; Jianping et al., 2004). However, the increase in DLS size is not due particle growth, but due to agglomeration. Thus, the suspensions are believed to be physically stable. Further stability tests could confirm this, i.e., both suspensions are physically stable for at least 3 month (data by now). Storing the hesperidin suspension for 30 days led to a decrease in the z-average from 717 to 518 nm and to a slight increase in PdI from 0.21 to 0.25. The observed decrease in size is probably due to the growth of larger crystals which sediment during analysis and thus cannot be detected by the instrument. Consequently only smaller particles remain in the measuring window, leading to a smaller detected particle size. The growth of larger crystals was confirmed by the LD measurements. The d(v) 0.50 increased from 0.94 mm to 1.11 mm. Thus, particles were found to experience slight Ostwald ripening. However, only slight increases were found for the d(v) 0.99, which increased from 4.35 mm to 4.63 mm. Therefore, it can be expected that the hesperidin nanosuspension will remain physically stable for a longer period of time. The most pronounced increase in size was found for the apigenin nanosuspension. The z-average increased from 262 nm to 302 nm, while the PdI decreased from 0.32 to 0.25. The increase in size and the decrease in the size distribution indicates Ostwald ripening, i.e., the growth of larger crystals at the expense of smaller ones. This is attributed to the presence of larger particles within the small sized main population. The results were expected because from all nanosuspensions produced, the apigenin nanosuspension possessed the smallest z-average after production but at the same time it possessed the largest d(v) 0.99 values. Hence, it possessed the broadest size distribution and was therefore predestinated to experience Ostwald ripening during storage. Therefore, to obtain a more stable suspension, the amount of larger particles needs to be reduced. As explained above, this can be achieved by optimizing the HSS process in regard to rotor–stator geometry, processing speed and time.
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4. Conclusion
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ARTcrystal1-technology combines HSS as pre-treatment step and HPH at reduced pressures. In this study, the HSS pretreatment step of the ARTcrystal1-technology was shown to be more effective than the classical pre-milling, typically applied for HPH. By applying the ARTcrystal1-process different flavonoid nanosuspensions could be produced. Particles obtained possess lower PdI values and mean sizes similar or smaller to suspensions produced by classical HPH. Similar to HPH, the diminution efficacy of the process was found to be strongly influenced by the properties of the raw materials. Larger crystals and needle like structures led to larger sizes of the final product. The process is faster and more economical than classical HPH or other
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combination techniques and thus can be seen as an alternative to conventional processes for the production of nanocrystals. Further optimization is required in regard to increase the efficacy of the HSS pre-treatment step to reduce larger remaining particles.
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Acknowledgement
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This project was supported by “Zentrales Innovationsprogramm Mittelstand” (ZIM), Förderkennzeichen: KF2161906CS2.
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References
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501 Al Shaal, L., Müller, R.H., et al., 2010. smartCrystal combination technology – scale 502 up from lab to pilot scale and long term stability. Die Pharmazie 65, 877–884. 503 Al Shaal, L., Shegokar, R., et al., 2011. Production and characterization of antioxidant 504 apigenin nanocrystals as a novel UV skin protective formulation. Int. J. Pharm. 505 420, 133–140. 506 Badve, M.P., Gogatea, P.R., et al., 2014. Hydrodynamic cavitation as a novel approach 507 for delignification of wheat straw for paper manufacturing. Ultrason. 508 Sonochem. 21, 162–168. 509 Chen, R., 2013. Tailor-made Antioxidative Nanocrystals: Production and in vitro 510 Efficacy. PhD Thesis. Free University of Berlin. 511 Deschner, E.E., Ruperto, J., et al., 1991. Quercetin and rutin as inhibitors of 512 azoxymethanol-induced colonic neoplasia. Carcinogenesis 12, 1193–1196. 513 Frömming, K.H.S., Frömming, J., 1993. Cyclodextrins in Pharmacy. Kluwer Academic 514 Publishers, Dordrecht. 515 Gao, L., Wang, E., et al., 2004. Microemulsion-directed synthesis of different CuS 516 nanocrystals. Solid State Commun. 130, 309–312. 517 Ghiasi, M., Heravi, M.M., 2011. Quantum mechanical study of antioxidative ability 518 and antioxidative mechanism of rutin (vitamin P) in solution. Carbohydr. Res. 519 346, 739–744. Jasinska, M., Baldyga, J., et al., 2014. Dispersion of oil droplets in rotor–stator mixers: Q5 520 521 experimental investigations and modeling. Chem. Eng. Process.: Process Intensif (in press). 522 Jianping, L., Longzhen, Q., et al., 2004. Controlled synthesis of magnesium hydroxide 523 nanoparticles with different morphological structures and related properties in 524 flame retardant ethylene–vinyl acetate blends. Nanotechnology 15, 1576–1581. 525 Keck, C.M., 2006. Cyclosporine Nanosuspensions: Optimised Size Characterisation & 526 Oral Formulations. PhD Thesis. Freie Universitaet Berlin. 527 Keck, C.M., 2010. Particle size analysis of nanocrystals: improved analysis method. 528 Int. J. Pharm. 390, 3–12. 529 Keck, C.M., 2011. Nanocrystals and amorphous nanoparticles and method for 530 production of the same by a low energy process. US 20130095198 A1. 531 Keck, C.M., Müller, R.H., 2006. Drug nanocrystals of poorly soluble drugs produced 532 by high pressure homogenisation. Eur. J. Pharm. Biopharm. 62, 3–16. 533 Keck, C.M., Müller, R.H., 2008. Size analysis of submicron particles by laser 534 diffractometry – 90% of the published measurements are false. Int. J. Pharm. 535 355, 150–163. 536 Mauludin, R., Müller, R.H., 2013. Physicochemical properties of hesperidin 537 nanocrystal. Int. J. Pharm. Pharm. Sci. 5, 954–960. 538 Mauludin, R., Müller, R.H., et al., 2010. Development of an oral rutin nanocrystal 539 formulation. Int. J. Pharm. 370, 202–209. 540 Mishra, P.R., Al Shaal, L., et al., 2009. Production and characterization of hesperetin 541 nanosuspensions for dermal delivery. Int. J. Pharm. 371, 182–189. 542 Müller, R.H., Gohla, S., et al., 2011. State of the art of nanocrystals – special features, 543 production, nanotoxicology aspects and intracellular delivery. Eur. J. Pharm. 544 Biopharm. 78, 1–9. 545 Petersen, R., 2008. Nanocrystals for use in topical cosmetic formulations and 546 method of production thereof, Google Patents. EP 2099420 A1. 547 Rodgers, T.L., Cooke, M., 2012. Correlation of drop size with shear tip speed. 14th 548 European Conference on Mixing, Warszawa. 549 Salazar, J., Müller, R.H., et al., 2014. Combinative particle size reduction technologies 550 for the production of drug nanocrystals. J. Pharm. 2014, 1–14. Scholz, P., Keck, C.M., 2014. Nanoemulsions produced by rotor–stator high speed Q6 551 stirring. Raw materials & packaging (submitted for publication). 552 Scholz, P., Arntjen, A., et al., 2014. ARTcrystal process for industrial nanocrystal 553 production – optimization of the ART MICCRA pre-milling step. Int. J. Pharm. 554 465, 388–395. 555 Wu, W., Nancollas, G.H., 1998. A new understanding of the relationship between 556 solubility and particle size. J. Solution Chem. 27, 521–531.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Pharmaceutical nanotechnology
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Flavonoid nanocrystals produced by ARTcrystal1-technology
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Patrik Scholz a,b , Cornelia M. Keck a,b, *
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a b
University of Applied Sciences Kaiserslautern, Applied Pharmacy Division, Campus Pirmasens, Germany Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 September 2014 Received in revised form 2 November 2014 Accepted 4 November 2014 Available online xxx
ARTcrystal1-technology is a novel technique for a more efficient production of nanocrystals. It consists of a high speed stirring (HSS) step as pre-milling and subsequent high pressure homogenization (HPH) at reduced pressure and cycle numbers. In this study, three antioxidants, rutin, hesperidin and apigenin, were processed by ARTcrystal1-technology, the results were compared to sizes obtained for the production of nanocrystals produced by classical HPH. By using the ARTcrystal1-process, all three substances could be transformed into nanosuspensions with mean sizes and PdIs of 431 nm/0.27, 717 nm/ 0.21 and 262 nm/0.31, respectively. Depending on the properties of the raw material the ARTcrystal1technology revealed similar or even better results than classical HPH. Further optimization of the setup of the HSS process might lead to an optimized process with higher efficacy than classical HPH. ã 2014 Published by Elsevier B.V.
Keywords: ARTcrystal Nanocrystal Rutin Hesperidin Apigenin
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1. Introduction
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Flavonoids, e.g., rutin, hesperidin and apigenin possess many positive biological effects. They scavenge reactive oxygen species, such as hydroxyl radicals and superoxide radicals, thus preventing biological and chemical substances from oxidative damage. These radicals can come into existence by UV-radiation. Furthermore, rutin has antiplatelet, antiviral and antihypertensive effects (Ghiasi and Heravi, 2011). Flavonoids have a light tumor growth inhibition potential (Deschner et al., 1991). Due to their manifold benefits, flavonoids have a great potential for supporting various disease treatments, thus being interesting substances in the pharmaceutical and nutraceutical field. However, a limiting factor is their poor solubility, restricting their full antioxidative potential as well as their bioavailability. To overcome this problem the solubility of the actives needs to be increased. During the last decades different ways to enhance the solubility of poorly soluble actives were developed. Examples are soluble drug salts, co-solvents or cyclodextrins (Frömming and Frömming, 1993). However, one of the most successful methods is the production of nanocrystals. Nanocrystals consist of 100% pure drug, only being stabilized by a small amount (1–2% (w/w)) of stabilizer and possess a mean crystal size below 1000 nm, typically between 200 and 500 nm (Keck and Müller, 2006). In contrast to
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* Corresponding author at: Fachhochschule/University of Applied Sciences Kaiserslautern, Campus Pirmasens – Applied Pharmacy, Carl-Schurz-Str. 10–16, 66953 Pirmasens, Germany. Tel.: +49 631 3724 7031; fax: +49 631 3724 7044. E-mail address: [email protected] (C.M. Keck).
larger crystals, nanocrystals possess a higher curvature. According to the Ostwald–Freundlich equation, this leads to a higher dissolution pressure and thus to a higher kinetic saturation solubility (Wu and Nancollas, 1998). Furthermore, nanocrystals possess a larger surface area. Thus, according to the Noyes–Whitney equation this leads to an increased dissolution velocity. Finally, the greater surface-to-mass ratio enables mucosal adherence of nanocrystals, allowing them to stay in the upper parts of the gastrointestinal tract until complete dissolution is ensured. As a consequence, the possibility of a fast gastrointestinal passage and fecal elimination of undissolved crystals is reduced and bioavailability is increased (Müller et al., 2011). In fact, the production of nanocrystals is the most elegant way to overcome poor solubility and to increase to bioavailability of poorly soluble actives. There are two main approaches of nanocrystal production, the bottom-up and the top-down approach. While the bottom-up process focuses on in situ forming of nanocrystals by precipitation, top-down processes produce nanocrystals by grinding large crystals into the nanoscale. Common top-down techniques are high pressure homogenization (HPH) and bead milling (Müller et al., 2011). Newer processes combine a pre-treatment step with subsequent HPH. Examples are freeze drying and HPH or bead milling and HPH (Salazar et al., 2014). These combination techniques aim at achieving smaller crystal sizes. Recently another combination process was introduced. This process is called the ARTcrystal1-technology and aims at a faster and more economic nanocrystal production compared to traditional top-down techniques. It combines an effective rotor–stator high speed stirring (HSS) process with subsequent HPH at reduced
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Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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numbers and cycles (Keck, 2011). Recent studies investigated the major process parameters of the HSS process, such as in process temperature and HSS pretreatment efficacy depending on raw material properties (Scholz et al., 2014) for the flavonoid rutin. To further prove the efficacy of the ARTcrystal1-technology, this study aimed at investigating the efficacy of the HSS process in comparison to the classical pre-milling step of HPH and to apply the method to different materials, i.e., to other flavonoids with different physicochemical properties. Finally, it was aimed at comparing the results obtained by ARTcrystal1-technology to the results obtained by classical HPH.
2. Materials and methods
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2.1. Materials
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Coarse rutin (Art.-No. 907861) and jet-milled rutin (Art.-No. 900313) were purchased from Denk Ingredients, Munich, Germany. Apigenin and hesperidin were purchased from Exquim, S.A., Barcelona, Spain. As stabilizer Plantacare1 2000 (decyl glucoside, BASF, Ludwigshafen, Germany) was used. Distilled water was obtained from a WDA 25 distillation unit (QVF PILOTTEC, Jena, Germany). All other chemicals were used as received.
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Fig. 1. DLS data (upper) and LD data (bottom) for coarse rutin processed by ARTcrystal1-technology compared to coarse rutin processed by the classical pre-milling step (rotor–stator mixing and HPH at low pressures).
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2.2. Methods
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In the first part of the study, the efficacy of the high speed stirring (HSS) step was compared to the efficacy of the classical pre-milling step of HPH. For this a coarse rutin suspension was pretreated by a classical high speed rotor–stator stirrer and premilled by HPH at low pressures. Results were compared to coarse rutin processed by ARTcrystal1-technology, i.e., effective rotor–stator high speed stirring and subsequent HPH at low pressures. In the second part, different flavonoids were processed by the ARTcrystal1-technology and the results were compared to results obtained by classical HPH. Finally the physical stability of the nanosuspensions was investigated to prove the absence of stability issues caused by Ostwald ripening or agglomeration.
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2.2.1. Preparation of macrosuspensions All suspensions contained 5% (w/w) drug material as well as 1% (w/w) Plantacare1 2000 as stabilizer and distilled water to up to 100.0%. Macrosuspensions were produced by dispersing the dry powder in the surfactant solution under magnetic stirring. The coarse suspensions were stored at 4 C in a refrigerator until use. 2.2.2. Production of nanosuspensions by ARTcrystal1-technology 1 liter of macrosuspension was processed in an ART MICCRA D27 rotor stator system (ART Labor- und Prozesstechnik, Müllheim, Germany). High speed stirring (HSS) as pretreatment prior to HPH was performed by using a rotor unit with a slit size of 1 mm and a stator with a slit size of 0.5 mm. The gap width between rotor and stator was 0.1 mm. Return flow to the product container was modulated by a valve position of 45 for reduction of foam formation (Scholz et al., 2014). HSS was performed for 5 min at 24,000 rpm. During the production the D27 system was cooled to 10 C using an Alpha RA12 cooling device (Lauda, LaudaKönigshofen, Germany). In-process temperature was controlled by a thermometer HI 8314 (HANNA Instruments, Kehl, Germany). After this HSS step, subsequently HPH was applied for 5 cycles at 300, 500 or 750 bar in an APV LAB 40 high pressure homogenizer (APV Deutschland GmbH, Germany). Cooling of the samples between the cycles was provided for stability reasons (Keck, 2006). 2.2.3. Production of suspensions by classical pre-milling of HPH Production, i.e., pre-milling, was performed as described in the literature (Mishra et al., 2009; Chen, 2013). Briefly, macrosuspensions were subjected to pre-mixing by using a rotor–stator dispersing unit (D9, ART Labor- und Prozesstechnik, Müllheim, Germany) at 39,000 rpm for 1 min. Subsequently, the suspension was subjected to HPH at low pressures, i.e., 2 cycles at 250 bar, followed by 5 cycles at 500 bar.
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2.2.4. Short-term physical stability Since stability issues are very important for pharmaceutical products, short term physical stability of the produced nanosuspensions was investigated for a period of 30 days when stored at room temperature.
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2.3. Particle size analysis
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Samples were analyzed regarding size, size distribution and morphology using dynamic light scattering, laser diffraction and light microscopy.
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particle size and the polydispersity index (PdI) as a measure for the width of the size distribution. Prior to the measurements, samples were diluted in saturated solutions of the given substance by a factor of 1:200. All measurements were performed at 25 C in triplicates. Results were analyzed by using the general purpose mode.
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2.3.2. Laser diffraction For laser diffraction (LD) a Mastersizer 2000 with Hydro S wet dispersion unit (Malvern Instruments, UK) was used. LD yields a volume based particle size distribution and is capable to analyze particles in the microscale as well as in the nanoscale size range. For particle population characterization, the volume-weighted diameters d(v) 0.50, d(v) 0.95 and d(v) 0.99 were chosen. All measurements were performed as triplicates in saturated drug
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Table 1 Particle size (LD data) and microscopic images (magnification 1000 fold) of the raw materials of coarse rutin, jet-milled rutin, hesperidin and apigenin. Material
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d(v) 0.50 d(v) 0.99 Microscopic image (1000 fold) (mm) (mm)
Rutin (coarse)
5.49
61.64
Rutin (jet milled)
10.50
129.16
4.96
86.73
55.90
150.23
Hesperidin
Apigenin
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2.3.1. Dynamic light scattering Dynamic light scattering (DLS) measurements were performed by a Zetasizer Nano ZS (Malvern Instruments, UK). DLS yields the intensity based hydrodynamic diameter (z-average) as mean Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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medium as dispersion medium (Keck, 2010). Mie theory was applied for size analysis by using the optical parameters (real/ imaginary refractive index) 1.593/0.02 for rutin (Keck, 2010), 1.59/ 0.01 for apigenin (Al Shaal et al., 2010) and 1.57/0.01 for hesperidin (Petersen, 2008). 2.3.3. Light microscopy Light microscopy was used to characterize particle morphology and to prove the results obtained by LD analysis (Keck and Müller, 2008; Keck, 2010). A DM1000 light microscope with MD170HD camera and Leica Application Suite software (Leica Microsystems, Wetzlar, Germany) was utilized for this purpose. 200 fold magnification was applied for overviewing the sample and detecting larger particles or agglomerates and 1000 fold
magnification was used for detailed characterization of the particles.
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3. Results and discussion
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3.1. Comparison of diminution efficacy of high speed stirring (HSS) vs. classical pre-milling
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Classical pre-milling was performed by rendering a coarse rutin suspension with a rotor–stator pre-mixing step (39,000 rpm, 1 min) and subsequent HPH (2 cycles at 250 bar, 5 cycles at 500 bar). ARTcrystal1-technology was applied by applying HSS for 5 min at 24,000 rpm, followed by 5 cycles of HPH at 500 bar. The results obtained are shown in Fig. 1.
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Fig. 2. DLS data (upper) and LD data (bottom) for coarse (left) and jet milled (right) rutin processed by ARTcrystal1-technology for up to 5 cycles at 300 bar and 500 bar.
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HSS was found to be more efficient than the classical premixing. Whereas the HSS led to a suspension with a d(v) 0.50 of 3.68 mm and a d(v) 0.99 of 28.6 mm (Fig. 1, left), the classical premixing, i.e., 39.000 rpm for 1 min, resulted in a d(v) 0.50 of 5.58 mm and a d(v) 0.99 of 125.85 mm (Fig. 1, right). Pre-milling for 2 cycles at 250 bar decreased the d(v) 0.50 to 3.18 mm and the d(v) 0.99 to 18.9 mm. Hence, about similar results were obtained by either applying HSS for 5 min or the classical pre-milling, e.g., 1 min stirring at 39,000 rpm and 2 cycles at 250 bar. Further processing at 500 bar HPH resulted in a further decrease in size for both suspensions. After 1 cycle at 500 bar no significant differences were obtained between the two samples. However, after 5 cycles at 500 bar samples processed with HSS were found to possess much smaller sizes and a narrower size distribution than the suspension processed with the classical pre-treatment.
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The HSS treated suspension led to a d(v) 0.50 of 0.39 mm, a z-average of 431 nm and a PdI of 0.27, whereas the suspension pretreated with the classical milling led to a d(v) 0.50 of 1.48, a z-average of 470 nm and a PdI of 0.41. Both suspensions contained larger remaining particles. However, the particles for the HSS pretreated suspension were distinctly smaller, than the particles which remained in the suspension pre-treated with the classical milling process. A possible explanation for the higher efficacy of the HSS might be that HSS was more efficient in destroying larger crystals and Q2 agglomerates. However, it might also be possible the HSS pretreatment created imperfections and damages in the crystal structures by hydrodynamic shearing. Hence, even though crystals could not be fully diminuted by HSS, they could be easily broken down by subsequent HPH. Classical pre-mixing at Q3
Table 2 Microscopic images of coarse and jet milled rutin: raw material prior to processing, after 5 min of HSS pretreatment and after 5 cycles HPH at 300 bar or 500 bar (magnification 1000). Jet milled rutin
Coarse rutin
Raw material
ARTcrystal1-pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 300 bar
ARTcrystal1 HPH 5 500 bar
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39,000 rpm by the D9 sowed only a small effect on particle size reduction due to turbulences inside the beaker, i.e., not every particle was forced to pass the rotor. Thus, even elongation of stirring would not result in a fine suspension. Additionally, the rotor inside the D9 is distinctly smaller, leading to less shear stress due to lower shear tip speed and thus reduced size reduction efficacy (Rodgers and Cooke, 2012). 3.2. Production of different flavonoid nanocrystals by ARTcrystal1technology To further investigate the nanonization potential of the ARTcrystal1-technology, different flavonoids with different physical properties, such as crystal size and morphology were processed. The aim was to determine the influence of different material properties, such as starting particle size and shape on
final crystal size after processing. Four flavonoid materials were selected and the raw material properties were analyzed by means of particle size and morphology. The results are shown in Table 1. As displayed in Table 1, the four raw materials differ in their properties. Coarse rutin contained mostly large monolithic, cuboid crystals with sizes of about 30 mm in length and of about 5 mm in diameter. Jet milled rutin raw material consisted mostly of very fine needles with submicron diameter and some large aggregates. Hesperidin raw material contained bundle-like shaped aggregates of thin needle-shaped crystals. Apigenin raw material consisted of very large crystals, as indicated by light microscopy and a d(v) 0.50 of 55.9 mm. Due to the different properties the flavonoids were found to be suitable to investigate if the ARTcrystal1-method is able to nanonize different raw materials with different physical properties, such as crystal size and morphology.
Fig. 3. DLS data (upper) and LD data (bottom) for hesperidin processed by ARTcrystal1-technology at 300, 500 and 750 bar.
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In the first study, coarse and jet milled rutin material were used to determine the influence of the size and crystal morphology of the starting material on the final product size. Results are shown in Fig. 2. 3.2.1. Coarse rutin Subjecting the coarse rutin suspension to HSS resulted in a reduction of the d(v) 0.99 from 61.6 mm to 28.6 mm after 5 min of stirring (Fig. 2, left). One cycle at 300 bar reduced the d(v) 0.99 of 21.4 mm, further processing resulted after 5 cycles in a d(v) 0.50 of 1.58 mm and a d(v) 0.99 of 9.57 mm. DLS measurements revealed a z-average of 471 nm and a PdI of 0.342, indicating the resulting suspension is in the submicron scale. However, the PdI was found to by >0.3. Thus, the suspension is very polydisperse. Increasing the pressure to 500 bar could further decrease the particle size. One cycle of HPH at 500 bar resulted in a d(v) 0.99 of 17.3 mm. Further processing led to a stronger decrease in particle size, especially in d(v) 0.99. After 5 cycles at 500 bar, the resulting nanosuspension possessed a z-average of 431 nm, a PdI of 0.272, a d (v) 0.50 of 0.39 and a d(v) 0.99 of 6.23 mm (Fig. 2, right). The resulting suspension still contained some microscale crystals, as proven by LD data and light microscopy (Fig. 2, Table 2). Nevertheless, the resulting suspension can be considered as a nanosuspension. 3.2.2. Jet milled rutin For the jet milled material, a strong reduction in size from 129.2 mm down to 4.5 mm (d(v) 0.99) within 5 min of HSS was obtained. This suspension already possessed nanoscale dimensions (572 nm, PdI of 0.29, Fig. 2, left). Subjecting this suspension after HSS pretreatment to HPH at 300 bar led to a further reduction of the d(v) 0.99 as well as to a reduction of the z-average. The resulting nanosuspension had a mean particle size of 488 nm, a PdI of 0.29 and a d(v) 0.99 of 3.37 mm. Additional cycles at 300 bar further reduced the crystal size slightly, mostly by destroying some of the remaining microcrystals. After 5 cycles at 300 bar, a nanosuspension with a z-average of 427 nm, a PdI of 0.21 and a d(v) 0.99 of 3.06 mm was obtained. The further reduction of PdI (from 0.29 after HSS to 0.21 after HPH at 300 bar) and d(v) 0.99 (from 4.54 mm to 3.06 mm) indicated an increase in homogeneity. Processing the pretreated suspension at 500 bar resulted in a small further decrease in size and a further slight increase in suspension homogeneity. After 5 cycles, a z-average of 411 nm, a PdI of 0.23 and a d(v) 0.99 of 2.84 mm could be achieved, revealing that 300 and 500 bar HPH led to nearly similar results. The differences in the results after processing coarse and jet milled rutin can be explained by light microscopy (Table 2). The jet milled material contained very large aggregates combined with a large amount of fine needles. The coarse material included nearly no aggregates, but large monolithic crystals. The fine needle aggregates can be destroyed with sufficient hydrodynamic shear forces, whereas the monolithic crystals need to be subjected to stronger forces, such as cavitation forces, in order to be broken down into crystals with desired sizes. Both, hydrodynamic shear forces and cavitation, occur in HPH (Keck and Müller, 2006), while particle breakdown in the high speed stirring is achieved mainly by hydrodynamic shear forces and minor cavitation forces (Badve et al., 2014; Jasinska et al., 2014). Thus, lager crystals require more energy input by HPH. Aiming at an increase in diminution efficacy of HSS, HSS at increased stirring rates (up to 36,000 rpm) or using rotors and stators with modified geometries and surfaces should be applied.
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nanosuspension (d(v) 0.50 1.84 mm, d(v) 0.99 7.49 mm, z-average 794 nm, PdI 0.54). Even HPH for 5 cycles at 500 bar was not sufficient to form a nanosuspension with a sufficient small size and size distribution (d(v) 0.50 of 1.49 mm, d(v) 0.99 of 5.84 mm, z-average 666 nm, PdI 0.34). To investigate if a higher energy input would lead to a smaller size, additional 5 cycles at 750 bar were
Table 3 Microscopic images of hesperidin: raw material prior to processing before processing, after 5 min of HSS pretreatment, and after 5 cycles of HPH at 300, 500 or 750 bar (magnification 1000). Raw material
ARTcrystal1 HSS pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 300 bar
ARTcrystal1 HPH 5 500 bar
ARTcrystal1 HPH 5 750 bar
3.2.3. Hesperidin After HSS the hesperidin microcrystals possessed a particle size distribution almost similar to the one of coarse rutin material (Fig. 3). The d(v) 0.50 was 3.34 mm and the d(v) 0.99 was 22.91 mm. Further processing for 5 cycles at 300 bar did not lead to a Please cite this article in press as: Scholz, P., Keck, C.M., Flavonoid nanocrystals produced by ARTcrystal1-technology. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.008
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applied. Indeed, the increase of the pressure to 750 bar resulted in a nanosuspension with a d(v) 0.50 of 0.94 mm, a z-average of 717 nm and a narrow PdI of 0.21. The efficient reduction in size was confirmed by light microscopy (Table 3). The raw material contained large aggregates of long rod-shaped crystals with a length of about 30 mm. After 5 cycles at 500 bar, the resulting nanosuspension contained only a small amount of microscale crystals (Table 3). In accordance to the results from the rutin nanosuspension, it can be concluded that HSS and subsequent HPH at 300 bar is not always powerful enough to form nanosuspensions from very coarse material. The coarse material contained large crystals of up to 100 mm, which could be broken down to around 22 mm after HSS. HPH at low pressures, such as 300 bar, seems not to be able to destroy these large crystals within 5 cycles. Increasing the energy input by processing 10 or 20 cycles and/or by increasing the homogenization pressure may result in nanosuspensions with smaller sizes. 3.2.4. Apigenin According to the results obtained from rutin and hesperidin, applying ARTcrystal1-technology with HPH at 300 bar pressure
did not seem suitable for raw materials consisting of mainly large crystals. Thus, for apigenin possessing a d(v) 0.50 of >50 mm (c.f. Table 1), the application of low pressure, i.e., 300 bar seemed to be not sensible, thus only pressures of 500 and 750 bar were applied. In contrast to hesperidin, after HSS for apigenin, a small d(v) 0.50 of 1.35 mm and a large d(v) 0.99 of 60.39 mm was found (Fig. 4). DLS results revealed a mean particle size of 327 nm and a PdI of 0.38. After only 1 cycle of HPH at 500 bar, the d(v) 0.50 was reduced to 0.48 mm, yet still large particles being present (d(v) 0.99 30.78 mm). After 5 cycles at 500 bar, the d(v) 0.99 was reduced to 8.43 mm with a z-average of 269 nm and a PdI of 0.33. Increasing the pressure to 750 bar, 5 cycles of HPH resulted in a nanosuspension with a d(v) 0.50 of 0.15 mm, a d(v) 0.99 of 6.21 mm, a z-average of 262 nm and a PdI of 0.318. Light microscopy shows the different material properties compared to hesperidin (Table 4). While hesperidin formed large bundle-shaped aggregates, apigenin raw material consisted of large, dense aggregates. Thus, it can be concluded that HSS and subsequent HPH are more efficient in diminuting larger crystals than agglomerates of needle like material. A possible explanation is the orientation of needles in
Fig. 4. DLS data (upper) and LD data (bottom) for apigenin processed by ARTcrystal1-technology at 500 and 750 bar.
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ARTcrystal1-pre-treatment (5 min at 24,000 rpm)
ARTcrystal1 HPH 5 500 bar
ARTcrystal1 HPH 5 750 bar
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the rotor–stator flow, allowing to bypass areas of high shear intensity due to their small diameter. To counteract this, a redesign of the rotor–stator geometry, e.g., by reducing the slit size, might enable to prevent particles from passing through areas of low shear intensity, thus leading to a more efficient reduction in size during the HSS pretreatment step.
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3.3. Comparison of ARTcrystal1-technology with classical HPH
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To estimate the efficacy of the Artcrystal-technology, the results obtained from this study were compared to results obtained by classical HPH. The results are summarized in Table 5. Compared to classical HPH (pre-milling and 20 cycles at 1500 bar), the ARTcrystal1-technology was found to lead to better results. By using the ARTcrystal-technology a coarse rutin nanosuspension with a size of 431 nm, a PdI of 0.27 and a d(v) 0.99 of 6.23 mm was produced, while classical HPH achieved a size of 727 nm with a PdI of 0.27 and a d(v) 0.99 of 2.40 mm (Mauludin et al., 2010). In fact, ARTcrystal-technology produced a nanosuspension with a distinctly smaller mean size while reaching the same homogeneity. However, the ARTcrystaltechnology was less sufficient in reducing the amount of remaining larger crystals. Jet milled rutin was processed by ARTcrystal1-technology to a nanosuspension with a size of 411 nm, a PdI of 0.23 and a d(v) 0.99 of 2.84 mm. Processing jet milled rutin by classical HPH resulted in a nanosuspension with a z-average of 381 nm and a very high PdI of 0.8 and a d(v) 0.99 below 1.5 mm. Hence, ARTcrystal1-technology formed a nanosuspension with improved homogeneity in comparison to HPH, discernable by the PdI values. The high PdI from HPH indicated agglomeration, which could not be detected by LD due to the influence of stirrer in the dispersion unit (Chen, 2013). Agglomeration is often caused by too high production temperatures which occur due to the high dissipation during HPH (Keck, 2006). In contrast, ARTcrystaltechnology only applies low pressures, thus less dissipation occurs during the production. Therefore, the ARTcrystal-process might be especially suitable for materials being sensitive to agglomeration during production. Hesperidin processed by ARTcrystal1 technology at 750 bar resulted in a nanosuspension with a size of 717 nm, a PdI 0.21 and a d(v) 0.99 of 4.35 mm. Processing of hesperidin suspensions, stabilized with either Poloxamer1 188, sodium dodecyl sulfate, Tween1 80 or polyvinyl alcohol, by HPH, resulted in crystal sizes of 239–449 nm, PdIs of 0.308–0.434 and d(v) 0.99 of 2.06–5.63 mm (Mauludin and Müller, 2013). While the mean particle size is larger compared to traditional HPH, a lower PdI could be achieved by ARTcrystal1-technology. Also these data indicate that the ARTcrystal process enables the production of suspensions with a narrower size distribution, when compared to classical HPH. However, the efficacy of destroying all larger particles seems to be slightly lower in comparison the classical HPH. These results were also confirmed by the results obtained for the apigenin nanosuspension. ARTcrystal1-technology produced a nanosuspension with a mean size of 262 nm, a PdI of 0.32 and a d(v) 0.99 of 6.21 mm. Classical HPH led to sizes of 484 nm, a PdI of 0.355 and a d(v) 0.99 of 2.78 mm (unpublished data). The data obtained from this study suggest to optimize the ArtCrystal process in regard to increase the efficacy in eliminating larger remaining particles. In case of classical HPH this optimization was achieved by combining bead milling and HPH (Petersen, 2008). This process is known as smartCrystal1 combination technique. Processing apigenin via this process led to a size of
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Table 5 Comparison of particle sizes obtained after production of nanosuspensions by ARTcrystal1-technology and classical HPH (data obtained from literature). Material
ARTcrystal1-technology (z-average, PdI)
Literature (z-average, PdI)
Rutin (coarse) Rutin (jet milled) Hesperidin Apigenin
431 nm, 0.27 411 nm, 0.23 717 nm, 0.21 262 nm, 0.32
727 nm, 0.27 (Mauludin et al., 2010) 381 nm, 0.8 (Chen, 2013) 239–449 nm, PdI 0.31–0.43 (Mauludin and Müller, 2013) 439 nm, 0.28 (Al Shaal et al., 2011)
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439 nm, a PdI of 0.28 and a d(v) 0.99 of 0.52 mm (Al Shaal et al., 2011). However, the smartCrystal1 process is time consuming, as it involves the bead milling as pre-treatment step. In contrast, the ArtCrystal process is extremely fast, i.e., it takes only a few minutes. Thus, if the efficacy of diminuting larger remaining crystals can be increased, the ArtCrystal process will be superior in comparison to classical approaches for the production of nanosuspensions. Earlier studies already indicated that elongation of the HSS stirring time might destroy remaining crystals with sizes above approximately 10 mm (Scholz et al., 2014). Alternatively, an increase of stirring speed to 36,000 rpm might allow faster and better destruction of larger particles. Further optimization potential lies in the optimization of the rotor and stator geometry, e.g., the reduction in the slit size (Scholz and Keck, 2014).
3.4. Short-term physical stability of the produced rutin nanosuspensions
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Since physical stability is an important issue a short term physical stability study was conducted. The aim of this stability study was to prove that the ARTcrystal1-technology is able to produce stable nanosuspensions. Results are displayed in Fig. 5. The results showed that the nanosuspensions were stable within 30 days. However, slight increases in size were observed for all suspensions. For both rutin nanosuspensions DLS data increased, whereas no changes were detected by LD measurements. Light microscopy revealed a slight agglomeration of the particles. The agglomerates were very loose and could be easily de-aggregated by light shacking. Therefore, these agglomerates could also be destroyed by light stirring in the wet dispersion unit of the LD instrument, thus
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Fig. 5. DLS data (top) and LD data (bottom) for short term physical stability (30 days) of flavonoid nanosuspensions (hesperidin, apigenin, jet milled and coarse rutin) processed by ARTcrystal1-technology.
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LD could not detect any change in particle size. However, DLS measurements are performed without stirring. Thus, during the DLS measurements no stirring induced hydrodynamic shear stress is applied to the sample, which consequently enables the detection of agglomerates. The agglomeration was more present in the jet milled suspension. A possible explanation for this is the needle like structure of the jet milled rutin, which possesses a greater surfaceto-mass ratio than the more cuboid crystals of the coarse rutin, thus they tend to agglomerate more easily (Gao et al., 2004; Jianping et al., 2004). However, the increase in DLS size is not due particle growth, but due to agglomeration. Thus, the suspensions are believed to be physically stable. Further stability tests could confirm this, i.e., both suspensions are physically stable for at least 3 month (data by now). Storing the hesperidin suspension for 30 days led to a decrease in the z-average from 717 to 518 nm and to a slight increase in PdI from 0.21 to 0.25. The observed decrease in size is probably due to the growth of larger crystals which sediment during analysis and thus cannot be detected by the instrument. Consequently only smaller particles remain in the measuring window, leading to a smaller detected particle size. The growth of larger crystals was confirmed by the LD measurements. The d(v) 0.50 increased from 0.94 mm to 1.11 mm. Thus, particles were found to experience slight Ostwald ripening. However, only slight increases were found for the d(v) 0.99, which increased from 4.35 mm to 4.63 mm. Therefore, it can be expected that the hesperidin nanosuspension will remain physically stable for a longer period of time. The most pronounced increase in size was found for the apigenin nanosuspension. The z-average increased from 262 nm to 302 nm, while the PdI decreased from 0.32 to 0.25. The increase in size and the decrease in the size distribution indicates Ostwald ripening, i.e., the growth of larger crystals at the expense of smaller ones. This is attributed to the presence of larger particles within the small sized main population. The results were expected because from all nanosuspensions produced, the apigenin nanosuspension possessed the smallest z-average after production but at the same time it possessed the largest d(v) 0.99 values. Hence, it possessed the broadest size distribution and was therefore predestinated to experience Ostwald ripening during storage. Therefore, to obtain a more stable suspension, the amount of larger particles needs to be reduced. As explained above, this can be achieved by optimizing the HSS process in regard to rotor–stator geometry, processing speed and time.
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4. Conclusion
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ARTcrystal1-technology combines HSS as pre-treatment step and HPH at reduced pressures. In this study, the HSS pretreatment step of the ARTcrystal1-technology was shown to be more effective than the classical pre-milling, typically applied for HPH. By applying the ARTcrystal1-process different flavonoid nanosuspensions could be produced. Particles obtained possess lower PdI values and mean sizes similar or smaller to suspensions produced by classical HPH. Similar to HPH, the diminution efficacy of the process was found to be strongly influenced by the properties of the raw materials. Larger crystals and needle like structures led to larger sizes of the final product. The process is faster and more economical than classical HPH or other
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combination techniques and thus can be seen as an alternative to conventional processes for the production of nanocrystals. Further optimization is required in regard to increase the efficacy of the HSS pre-treatment step to reduce larger remaining particles.
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Acknowledgement
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This project was supported by “Zentrales Innovationsprogramm Mittelstand” (ZIM), Förderkennzeichen: KF2161906CS2.
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References
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