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Development of Curcumin Nanocrystal: Physical Aspects
Pharmaceutical Nanotechnology Development of Curcumin Nanocrystal: Physical Aspects 2 ¨ HENI RACHMAWATI,1,2 LOAYE AL SHAAL,2 RAINER H. MULLER, CORNELIA M. KECK2 1
Pharmaceutics Research Group, School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia
2 Freie Universit¨at Berlin, Department of Pharmacy, Pharmaceutical Technology, Biopharmaceutics & NutriCosmetics, Berlin, Germany
Received 23 June 2012; revised 25 August 2012; accepted 14 September 2012 Published online 9 October 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23335 ABSTRACT: Curcumin, a naturally occuring polyphenolic phytoconstituent, is isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae). It is water insoluble under acidic or neutral conditions but dissolves in alkaline environment. In neutral or alkaline conditions, curcumin is highly unstable undergoing rapid hydrolytic degradation to feruloyl methane and ferulic acid. Thus, the use of curcumin is limited by its poor aqueous solubility in acidic or neutral conditions and instability in alkaline pH. In the present study, curcumin nanocrystals were prepared using high-pressure homogenization, to improve its solubility. Five different stabilizers [polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), D-"-tocopherol polyethylene glycol 1000 succinate (TPGS), sodium dodecyl sulfate (SDS), carboxymethylcellulose sodium salt] possessing different stabilization mechanism were investigated. The nanoparticles were characterized with regard to size, surface charge, shape and morphology, thermal property, and crystallinity. A short-term stability study was performed storing the differently stabilized nanoparticles at 4◦ C and room temperature. PVA, PVP, TPGS, and SDS successfully produced curcumin nanoparticle with the particle size in the range of 500–700 nm. PVA, PVP, and TPGS showed similar performance in preserving the curcumin nanosuspension stability. However, PVP is the most efficient polymer to stabilize curcumin nanoparticle. This study illustrates that the developed curcumin nanoparticle held great potential as a possible approach to improve the curcumin solubility then enhancing bioavailability. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:204–214, 2013 Keywords: curcumin; nanocrystals; steric stabilization; electrostatic stabilization; polymers; high-pressure homogenization; surface charge; aggregation; crsytallinity; nanoparticle
INTRODUCTION Curcumin, a naturally occuring polyphenolic phytoconstituent, is isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae). Curcumin is chemically (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1, 6-diene-3,5,-dione. It has a pKa1 , pKa2 , and pKa3 value of 7.8, 8.5, and 9.0, respectively, for three acidic protons.1,2 It is insoluble in water under acidic or neutral conditions but dissolves in alkaline environment. Curcumin is highly unstable undergoing rapid hydrolytic degradation in neutral or alkaline conditions to feruloyl methane and ferulic acid.3 Thus, the use of curcumin is limited by its poor Correspondence to: Heni Rachmawati (Telephone: +62-222504852; Fax: +62-22-2504852; E-mail: [email protected], h [email protected]) Journal of Pharmaceutical Sciences, Vol. 102, 204–214 (2013) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
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aqueous solubility in acidic or neutral conditions and instability in alkaline pH. On pharmacological evaluation, curcumin was found to posses anticancer,4,5 antioxidant,6,7 antiinflammatory,8,9 hyperlipidemic,10,11 antibacterial,12 wound-healing,13 and hepatoprotective14,15 activities. The pharmacological efficacy of curcumin makes it a potential compound for treatment and prevention of a wide variety of human diseases. In addition, it is extremely safe upon oral administration even at very high doses, as proven in various animal models or human studies.16–18 Despite this, curcumin has not yet been approved as a therapeutic agent. The relative bioavailability of curcumin has been highlighted as a major problem for this. Curcumin is slightly absorbed in the gastrointestinal tract with the maximum solubility to be 11 ng/mL in plain aqueous buffer pH 5.0. The oral bioavailability of curcumin is very low (1% in rat). In clinical trial, quantifiable serum levels of
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curcumin were not achieved after oral administration of higher dose of curcumin (8 g/day).17 Attempts have been made to improve the solubility of curcumin by its chemical derivatization, complexation, or interaction with macromolecules, for example, gelatin, polysaccharides and proteins, and cyclodextrins. However, limited complexation, high-molecular weight of cyclodextrins, and pH of the processing medium limit their practical utility. Consequently, research still focuses on the development of formulation approaches to overcome the oral bioavailability problem. Ideal would be a universal formulation practically applicable to any drug. The drug nanocrystals are such a universal formulation approach. The special properties of drug nanocrystals are increased saturation solubility and increased surface area, both leading to an increase in the rate of dissolution and subsequently bioavailability. The nanocrystal approach can be used for all drugs for which the dissolution velocity is the rate-limiting step for absorption/bioavailibility, as for curcumin. The major production methods for drug nanocrystals are pearl milling and high-pressure homogenization (HPH),19,20 which were used here to produce curcumin nanocrystals. The stabilization ability of the stabilizers is a critical parameter determining the minimal achievable size and subsequent physical stability. The physical stability of nanocrystals is essential because aggregated nanocrystals loose the increased dissolution velocity. However, different drugs require different stabilizers. The stabilization ability of four typical sterically stabilizing polymers, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), D-"-tocopherol polyethylene glycol 1000 succinate (TPGS), and carboxymethylcellulose sodium salt (Na-CMC), were investigated. In addition, a lowmolecular weight surfactant, sodium dodecyl sulfate (SDS) was used as comparison for an electrostatic stabilizer. The obtained nanocrystals were characterized in term of size and particle charge [zeta potential (ZP)]. To examine the physical stability, a short-term stability study was performed storing the differently stabilized nanosuspensions at two different temperatures.
MATERIALS AND METHODS Materials Curcumin extract from Curcuma xanthorrhiza rhizome (purity 95%) was supplied by PT Phytochemindo Reksa (Bogor, Indonesia). The stabilizers PVA (MW = 31 K; Fluka Chemica GmbH, Steinheim, Germany), PVP (MW = 25 Kda; Sigma–Aldrich Chemie GmbH, Steinheim, Germany), TPGS (MW = 1513 da; Eastman Chemical Company, Liverpool, UK), Na-CMC (MW = 90 Kda; Sigma–Aldrich Chemie), and SDS DOI 10.1002/jps
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(MW = 288 da; Fluka Chemie GmbH) were used in this study. Ultra purified water (milli-Q; Millipore GmbH, Darmstadt, Germany) was used for all formulations. The water conductivity used for ZP analysis is 50 :S/cm. The other chemicals were of analytical reagent grade. Methods
Preparation of Nanosuspensions Nanosuspensions with different stabilizers were produced by HPH using a Micron Lab 40 (APV Deutchland GmbH, Unna D-59425, Germany) with a batch size of 40 mL. The best formulation of nanosuspensions for homogenization process contained 5.0% (w/ w) curcumin and 1.0% (w/w) of each stabilizer, except for SDS, which was 0.2% (w/w). Before the homogenization, the dispersion medium was prepared by dissolving the stabilizer in water. Curcumin powder was poured into the aqueous stabilizer solution under magnetic stirring (IKA-combimag RCT; IKALabortechnik, Staufen 79219, Germany) for 2 min. After dispersion, the suspension was then transferred into the bulk container of the homogenizer and homogenization was run with two cycles at 300, 500, and subsequently 1000 bar, as premilling steps. Afterward, HPH at 1500 bar was conducted applying 20 cycles. The progress of homogenization was controlled by size analysis of samples taken after 1, 5, 10, 15, and 20 homogenization cycles.
Particle Size Analyses Photon correlation spectroscopy (PCS; Zetasizer Nano ZS; Malvern Instruments, Malvern, UK) and laser diffractometry (LD; Coulter LS 230; BeckmanCoulter, Krefeld 47807, Germany) were employed to determine the particle size of curcumin nanosuspensions. PCS yields the mean diameter of the bulk population (z-average) and the polydispersity index (PI) as measure for the width of the size distribution. The measuring range of the zetasizer is approximately 0.6 nm to 6 :m. Larger particles in the micrometer range were therefore measured with LD yielding volume-weighted diameters. Analysis of the diffraction patterns was performed using the Mie theory, with a dispersant refractive index of 1.33, a particle refractive index with a real part of 1.50, and an imaginary part of 0.01. The results are given as diameter d(v)50%, d(v)95%, and d(v)99% values, representing the percentage of particles below given size (:m).
ZP Determination The ZP of the nanosuspensions was analyzed directly after production using the Zetasizer Nano ZS (Malvern Instruments). For this purpose, the zetasizer was switched from PCS mode to the laser doppler anemometry mode. The measurement is a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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particle electrophoresis, the particle velocity is determined via the Doppler shift of the laser light scattered by the moving particles. The field strength applied was 20 V/cm. The electrophoretic mobility was converted to the ZP in mV using the Helmholtz– Smoluchowski equation. To study different aspects of the adsorption of ionic and nonionic stabilizers, different measuring media were applied (distilled water and original dispersion media). Bidistilled water having its conductivity adjusted to 50 :S/cm with 0.9% NaCl solution was used to determine the Stern potential being directly correlated to the surface charge of the nanocrystals (Nernst potential). Using a standard conductivity adjusted with 1:1 electrolyte avoids fluctuation in the ZP because of the variations in the conductivity of distilled water especially when the sample itself contains electrolytes when adding it to the distilled water. In addition, the ZP was also measured in the original media of the nanosuspension as a measure of the thickness of the diffuse layer. The higher the measured ZP in these conditions, the thicker the diffuse layer and the more stable is the suspension. On the basis of the ZP data, long-term stability can be estimated.
Microscopic Analysis A Leitz-Orthoplan (Wetzlar, Deutchland) microscope was used. Samples were analyzed with polarized light at 160-fold magnification to obtain an overview information of the whole system and for the detection of crystals or aggregates >1 :m.
Nanocurcumin Morphology The morphology of PVP-nanocurcumin was observed using scanning electron microscope (SEM). SEM was performed on nanocurcumin produced using PVP as a stabilizer, which is appointed as the most appropriate polymer for this study. The samples were fixed on a brass stub using double-sided tape and then gold coated in vacuum by a sputter coater. The pictures were taken at excitation voltage of 10 kV and at 10,000–40,000× magnification by using JSM-6360LA Scanning Microscope (Jeol, Tokyo, Japan).
Thermal Property of Nanocurcumin The thermal characteristic of curcumin and PVPnanocurcumin was analyzed using differential thermal calorimetry (DSC). The DSC thermogram of curcumin and PVP-nanocurcumin were recorded using a differential scanning calorimeter (NETZSCH STA 449F1 STA449F1A-0164-M). Approximately 10 mg of each sample was heated in an open aluminum pan from 20◦ C to 300◦ C at scanning rate of 10◦ C/min under a stream of nitrogen. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
Crystallinity of Nanocurcumin The crystalline phase of both curcumin and PVPnanocurcumin were determined by X-ray powder diffraction (XPRD) by using a Diano (Woburn, Massachusetts) diffractometer with Cu-K" radiation (λ = 0.1540 nm). A scan rate of 0.04◦ /s was applied to record the pattern in the 2θ range of 20◦ –70◦ .
Stability Test of the Nanosuspension To investigate the effect of the stabilizers on the physical stability of the nanosuspensions, a short-term stability test was performed. The nanosuspensions were stored in sealed vials at different temperatures [room temperature (RT) and 4◦ C] for 30 days. Samples were taken on day 0 (day of production), day 7, and day 30. Characterization was carried out regarding particle size analyzed by PCS, LD, and polarized light microscopy.
RESULTS AND DISCUSSION Particle Size and Size Distribution as a Function of Homogenization Cycles and Type of the Stabilizer Nanoparticles are inevitably more unstable than microparticles because of the extra Gibbs free energy contribution related to the particle size and primarily due to the surface energy.21 A careful evaluation of the type and concentration of the stabilizer used is the key to the production of physically stable nanosuspensions. Five formulations of nanosuspension were prepared in this study with 1% (w/w) of PVA, PVP, Vitamin E TPGS, Na-CMC, and 0.2% (w/w) of SDS as stabilizers, respectively. In case of polymers used in the preparation of nanocrystals, these molecules should adsorb onto the surfaces of drug nanocrystals and provide steric stabilization effect.22 When their concentration is very low, it is often the case that the polymers bridge the particles and the dispersion is destabilized. According to our experience, the dispersion is usually stabilized by increasing the polymer concentration to the order of 1%. This is because most of the surfaces of the particles are covered by the adsorbed polymers. The adsorbed chain molecules on surface have ceaseless thermal motion, resulting in dynamically rough surface preventing coalescence by repulsive entropic force. Preliminary study was performed to optimize the concentration of each stabilizer, based on the process feasibility. The curcumin crystalline powder as received had LD d(v)50% of 5.89 :m and d(v)99% of 105.99 :m. The influence of the homogenization cycles on the particle size and size distribution is shown in Figure 1. After 20 cycles, smallest size was obtained for the TPGS stabilized system, the size increased in the order of: TPGS < SDS < PVP < PVA < Na-CMC. DOI 10.1002/jps
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Figure 1. Particle size (PCS, z-average) and width of the size distribution (polydispersity index, PI) as function of homogenization cycles for the five different curcumin nanosuspensions. (PM = values after premilling.)
It was described that the resulting final nanocrystal size does not depend on the type of stabilizer.23,24 However, different sizes were obtained by different types of stabilizers. As expected, SDS as well dispersing, fast diffusing low-molecular weight surfactant should reveal the best performance in the nanoparticle production. SDS-stabilized suspension effectively produced nanoparticle after five homogenization cycles with average size of particle about 630 nm and a PI of 0.56. Further homogenization cycles only changed a minor size reduction with steady in the particle distribution. However, TPGS exerted even better effectivity in producing crystals being in the nanometer range with narrow distribution just after premilling. Further homogenization cycles, that is, after 10 cycles, only slightly decreased the particle size and the steady state value was achieved with the size of about 550 nm and a PI of 0.47. This result can be explained by considering the chemical structure of TPGS. TPGS comprises amphiphilic properties, with a hydrophilic–lipophilic balance of about DOI 10.1002/jps
13. Moreover, its hydrophilic tail (polyethylene glycol) and hydrophobic portion (tocopherol succinate) are bulky and have large surface areas. Such characteristic makes it a good stabilizer. The performance of TPGS can be explained by the combination of low viscosity and high surface activity.25,26 This is a characteristic of advantages, which makes TPGS superior to other stabilizers used in this production. The ability to quickly cover depends on the molecular weight of the surfactants (diffusion velocity to the particle surface). The lower the molecular weight of surfactants, the faster they diffuse and vice versa. This was confirmed in this study. The higher the molecular weight of polymers (in case of PVA and Na-CMC), the slower was the decrease in particle size with increasing number of cycles (Fig. 1). For that reason, PVP with lower molecular weight than PVA and Na-CMC exhibits faster size reduction and stabilization. Na-CMC showed the least efficient size reduction. Smallest size (824 nm) was reached after 15 cycles. At 20 cycles, the size increased to 878 nm. This can be explained that a too JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 2. Particle diameter d(v)50%, d(v)95%, and d(v)99% (LD data) as a function of homogenization cycles for the five different curcumin nanosuspensions.
high input of energy in the homogenization process leads to aggregation in suspensions.27 The larger PCS sizes of the polymer-stabilized nanocrystals can be explained by aggregation due to the polymer bridging. The steady decrease in PI as shown in Figure 1 for all nanosuspensions confirms that at higher cycles mainly remaining larger particles are removed, and only limited further size reduction of the bulk population takes place. The influence of different stabilizers in producing curcumin nanosuspension can also be seen from LD data (Fig. 2). There is no change in the diameter d(v)50% characterizing the bulk population when moving from cycle 10 to 20, for all nanosuspensions. Only Na-CMC-stabilized nanosuspension showed significant reduction in d(v)95% when the homogenization cycle was increased from 15 to 20. A further more or less pronounced decrease was observed for all nanosuspensions in the diameter d(v)99%, being a sensitive measure of the presence of even a few larger particles. According to the LD data, again TPGS exerted the best stabilizing performance, whereas Na-CMC is the least efficient. The main reason for this different ability in the formation and stabilization of generated nanocrystals appears to be the stabilizer nature. The choice of stabilizer is therefore specific to each drug candidate and each formulation procedure. To preserve the nanocrystals from aggregation, the stabilizer should exhibit sufficient affinity on the particle surface. When Na-CMC with molar mass of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
90 Kda was used, the particle diameter of the resulting dispersion is larger. This means the addition of an anionic polyelectrolyte of high molar mass increases the tendency to form larger particle aggregates, attributed to polymer bridging. It should provide better stabilization than PVP or PVA because in Na-CMC steric and electrical stabilization principles are combined. ZP Determination The values of ZP of five differently stabilized nanosuspensions are presented in Table 1. In regard to the successful nanocrystal production reflected in the PCS and LD profiles above (Figs. 1 and 2), there is a close relationship between the ZP value and the size characteristic of obtained nanocrystals. SDSstabilized nanosuspension has the highest ZP value Table 1. Zeta Potential Values of the Five Differently Stabilized Curcumin Nanosuspensions; Left: Analyzed in Water (Conductivity 50 :S/cm, pH 5.8); Right: Analyzed in the Original Dispersion Medium
Stabilizer
Analyzed in Water (mV)
Analyzed in Original Dispersion Medium (mV)
Conductivity (:S/cm)
PVA PVP TPGS SDS Na-CMC
−5.5 −15.9 −12.5 −30.6 −26.6
−2.3 −11.9 −15.9 −52.2 −37.7
131 40 9 2640 1849
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Figure 3. Scanning electron microscopic photograph of curcumin (a) and PVP-nanocurcumin (b). Magnification 40,000×.
(−30.6 in water and −52.2 in SDS solution) among others even at low concentration (0.2%, w/w). SDS, an excellent electrostatic stabilizer, adsorbs the negatively charged alkyl chain of the molecule with high affinity onto particle surfaces leading to a high ZP. This high ZP of SDS is in accordance to the PCS diameter, in which after five cycles of homogenization, the most pronounced decrease took place. Both particle size and distribution remained unchanged up to cycle 20. This confirms that aggregate formation during the homogenization process—input of more energy is applied—can be avoided by the presence of SDS on the surface. The higher ZP of SDS-stabilized nanosuspension measured in original dispersion medium (SDS solution) is in agreement with the ZP theory. The negatively charged part of the molecule (alkyl chain with SO4 2− ) was adsorbed onto the particle surface and leading to an inner Helmholtz adsorption layer more densely packed than in distilled water. Adsorption takes place according to the theory of adsorption isotherms.28 Consequently, the potential of the inner Helmholtz layer increases leading subsequently to an increase of the ZP. As an ionic high-molecular weight polymer, Na-CMC provides a combination of electrostatic and steric stabilizations. An increased adsorption can also explain the increase of the ZP value of Na-CMC-stabilized nanosuspension when measured in the Na-CMC solution. The increase of the ZP in the original dispersion medium is similar high as the observed increase in SDS-stabilized nanosuspension (approximately by 20 mV). The increase of the potential of the inner Helmholtz layer due to the increased adsorption of negatively charged carboxylic (COO− ) groups onto particle surface has obviously more predominant effect than the shift of plain of shear due to the adsorption layer of the polymer chain. In addition, the negative charges of the Na-CMC are in but also on the surface of the polymer layer, thus increasing the Stern and subsequently ZP. The net result is therefore an increase in ZP from −26 to −37 mV. The ZP is higher than 30 mV, that is, above the limit required DOI 10.1002/jps
for physically stable dispersion systems.28 The ZP is therefore not the reason why nanosuspension production using Na-CMC was less effective compared with the SDS (Figs. 1 and 2). This supports the assumption that the aggregate formation was caused by bridging. In contrast, nanosuspensions with steric stabilizers (PVA, PVP, and TPGS) exhibited lower ZP values in water (−5.5, −15.9, and −12.5 mV, respectively). The adsorption layer of these polymers shift the plain of shear, at which the ZP is measured, to a larger distance from the particle surface. Consequently, the measured ZP is lower. The measured ZP values for the three nonionic steric stabilizers are similar in original dispersion medium and in distilled water. This indicates no desorption when the nanosuspensions are diluted with water for the measurement. This can be explained by the multipoint attachment of adsorbed polymers. In contrast the low-molecular weight, end-on (= single point) attached SDS is desorbed, the ZP decreases from about −52 to −31 mV. Of course, Na-CMC is also a polymer with ability to multipoint attachment. However, binding to the surface is less firm compared with the nonionic polymers. The repulsion between the negatively charged nanocrystal surface and the negatively charged CMC chains weakens the adsorptive forces, leading to partial desorption upon dilution. The measured ZP decreases. However, it does not indicate that these nanosuspensions exhibiting low physical stability as the stabilization occur through a steric mechanism. Thus, a reduction of the measured ZP is not necessarily correlated with a reduced stability. As the particle charge is one of the factors determining the physical stability of dispersion system, the ZP value can be used to predict the physical stability of these suspensions. Considering the obtained ZP results, it could be judged that all the investigated stabilizers should be suitable for the stabilization of curcumin nanosuspension. However, Na-CMC was not being a nice example for the destabilizing effect of polymer interactions. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 4. DSC of curcumin (a) and PVP-nanocurcumin (b).
Morphology of PVP-Nanocurcumin
Thermal Characteristic of PVP-Nanocurcumin
Figure 3 shows the morphologies of curcumin and PVP-nanocurcumin with aggregated status. Spherical morphology with a highly porous, foamlike structure can be observed only in curcumin nanoform. Particle size of the nanoparticles was drastically decreased from microscale to nanoscale (±400 nm).
Differential thermal calorimetry detects phase transition: glass transition (exothermic), crystallization, and (endothermic) melting. DSC thermograms were obtained to define the physical state of the drug and polymer in the nanoparticles and to detect any drug polymer interactions within the PVPnanocurcumin. Typical thermograms of curcumin and
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Figure 5. XPRD of curcumin (red line) and PVP-nanocurcumin (blue line).
PVP-nanocurcumin were shown in Figure 4. The purpose of these analyses was to detect possible alteration in the structure and nature of compound due to their organization in the form of nanoparticle. The DSC curve of curcumin as well as its nanoform showed a single endothermic peak at 180.5◦ C and 173.9◦ C, respectively. These results suggest that the homogenization process did not change the crystallinity of curcumin. The slightly changes in the melting temperature may be due to the presence of PVP in the mixture as also confirmed by other study.29 Crystallinity of PVP-Nanocurcumin Crystallinity characteristic are analyzed with XPRD when diffraction pattern of the X-ray from the sample is determined as a function of scattering angle.30 XPRD analysis of curcumin and PVP-nanocurcumin were performed and are illustrated (Fig. 5). No other peaks were observed, which is indicating the high purity of the samples. The XPRD data clearly explained that after homogenization, the nanocurcumin remains in the crystalline state although with less intensity. This seems to be in line with the DSC data, which suggests any slightly alteration in the crystallinity. The low percentage of PVP in the formulation (1%) seems not significantly interfering the diffractogram of PVP-nanocurcumin. Short-Term Stability Test of Curcumin Nanosuspension Although the number of articles describing nanosuspension formulations is extensive, limited literature DOI 10.1002/jps
is currently available that aims to evaluate and compare the ability of different stabilizers in their stabilizing potential from a more fundamental point of view, as reported here. The manufacturing of a nanosuspension implies the creation of additional surface area and hence interface. The nanosuspensions formed are thermodynamically less stable and tend to minimize their total energy by agglomeration. Kinetically, the process of agglomeration depends on its activation energy, which can be influenced by the presence of stabilizers to the system.20 To evaluate the stabilizing potential of stabilizers during storage, a stability test for 30 days was performed at two different temperatures (25◦ C and 4◦ C). The parameters observed in this study are particle size (Fig. 6) and polarized light microscopy (Fig. 7), performed after the nanosuspensions were stored for 7 and 30 days. The effect of different stabilizers on the physical stability of nanosuspensions is reflected by the PCS mean diameter and the LD diameter d(v)95% and d(v)99% (Fig. 6). The sterically stabilized nanosuspensions (PVA, PVP, and TPGS) exhibited a good performance in preventing particles from agglomeration at both temperatures. No or little changes in LD and PCS diameters were detected, except for PVA-stabilized nanosuspension minor, negligible increased in PCS diameter was observed. In accordance with the PCS and LD data, the polarized micrograph revealed no detectable crystal growth or agglomeration (Fig. 7). To summarize, the nanosuspensions JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 6. PCS diameter and LD diameter d(v)95% and d(v)99% of five differently stabilized curcumin nanosuspensions as function of days (0–30) stored at room temperature (RT, left) and at 4◦ C (right).
stabilized with PVA, PVP, and TPGS are stable over period of 30 days at both storage conditions. On the basis of the ZP data and the small sized obtained, SDS-stabilized nanosuspension was expected to be physically stable. However, even a marked increase both of d(v)95% and d(v)99% was observed. The increase in LD diameter indicates agglomeration during the storage. The formed agglomerates were not dispersed by the instrument stirring in the sample cell during LD measurement. This indicates strong interactive forces in the agglomerates because loose agglomerates are dispersed by dispersing forces in the sample cell. Moreover, polarized light micrograph (Fig. 7) clearly proved that after 7 and 30 days of storage, agglomerations took place. The decrease in PCS size can be explained that small sized aggregates (a few :m) continued to agglomerate increasing in size leaving the measuring range of PCS. The small sized, more stable, and nonaggregated nanocrystals remained. It is indeed contradictive with the ZP value, which is actually in the range JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
of good stability (>30 mV). The potential reason to explain this situation is that SDS formed a double layer on the particle surface, which hydrophobized the surface. This promotes hydrophobic interaction leading to particle aggregation. In contrast with the nanosuspension stabilized by Na-CMC, a significant increase in PCS diameter in time was observed, especially when stored at RT. However, no change in LD size was detected. The relatively constant diameter observed in LD measurements due to the formation of loose aggregates. They were easily deaggregated by stirring during the measurements as observed previously for hesperetin nanosuspensions [not published yet]. This argument is confirmed by the polarized micrograph (Fig. 7), which clearly showed agglomerate growth over 7 and 30 days of storage. PCS measurements showed an increase in the PCS diameter, attributed to slow continuing agglomeration of nanocrystals in the measuring range of PCS. Deagglomeration did not take place during the measurement because the samples are not DOI 10.1002/jps
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lize the produced crystals in the concentration used. PVA, PVP, and TPGS showed similar performance in preserving the curcumin nanosuspension stability. However, for further pharmaceutical processing reason, PVP is suggested as the most efficient polymer to stabilize curcumin nanocrystal. Oral curcumin nanocrystals appear as a potential natural product in the forefront of therapeutic agents for treatment of human diseases.
ACKNOWLEDGMENTS This project was financially supported by DAAD (German Academic Exchange Service), under scheme of Indonesian–German Scientists Exchange Program.
REFERENCES
Figure 7. Polarized light micrographs (magnification 160×) of the nanosuspensions after 7 and 30 days of storage at RT (7D and 30D). Nanosuspension stabilized with nonionic polymers (PVA, PVP, and TPGS) are stable with no agglomeration detected, in contrast to ionic stabilized nanosuspensions (SDS and Na-CMC).
stirred. Considering these results, it can be concluded that Na-CMC is not sufficient to stabilize aqueous curcumin nanosuspensions.
CONCLUSIONS Nanocrystals of curcumin were succesfully produced with four stabilizers PVA, PVP, TPGS, and SDS with the particle size in the range of 500–700 nm. Na-CMC resulted in an only slightly larger PCS diameter (about 800 nm) but distinctly higher diameter d(v)99%, indicating presence of aggregates. This indicates that this polymer is not able to efficiently stabiDOI 10.1002/jps
1. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. 2007. Biovailability of curcumin: Problems and Promises. Mol Pharm 4(6):808–818. 2. Tonnesen HH, Masson M, Loftsson T. 2002. Studies of curcumin and curcuminoids, cyclodextrin complexation, solubility, chemical and photochemical stability. Int J Pharm 244:127–135. 3. Wang YJ, Pan MH, Chery AL, Lin LI, Hoy S, Hsieh CV, Lia JK. 1997. Stability of curcumin in buffer solution and characterization of its degradation product. J Pharm Biomed Anal 15:1867–1876. 4. Ruby AJ, Kuttan G, Babu KD, Rajasekharan KN, Kuttan R. 1995. Antitumor and antioxidant activity of natural curcuminoids. Cancer Lett 94:79–83. 5. Gescher AJ, Sharma RA, Steward WP. 1992. Cancer chemoprevention by dietary constituents: A tale of failure and promise. Lancet Oncol 11:226–230. 6. Jayaprakasha GK, Jaganmohan L, Sakariah KK. 2006. Antioxidant activities of curcumin, demethoxy curcumin and bisdemethoxy curcumin. Food Chem 98:720–724. 7. Tonnesen HH, Greenhil JVL. 1992. Studies on curcumin and curcuminoids XXII curcumin as a reducing agent and as a radical scavenger. Int J Pharm 87:79–87. 8. Chan MM, Huang HI, Fenton MR, Fong D. 1998. In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties. Biochem Pharmacol 55(12):1955–1962. 9. Kim YS, Ahn Y, Hong MH, Joo SY, Kim KH, Sohn IS, Park HW, Hong YJ, Kim JH, Kim W, Jeong MH, Cho JG, Park JC, Kang JC. 2007. Curcumin attenuates inflammatory responses of TNF-alpha-stimulated human endothelial cells. J Cardiovasc Pharmacol 50(1):41–49. 10. Vanisree AJ, Sudha N. 2006. Curcumin combats against cigarette smoke and ethanol-induced lipid alterations in rat lung and liver. Mol Cell Biochem 288(1–2):115–123. 11. Rukkumani R, Aruna K, Varma PS, Rajasekaran KN, Menon VP. 2005. Comparative effects of curcumin and its analog on alcohol- and polyunsaturated fatty acid-induced alterations in circulatory lipid profiles. J Med Food 8(2):256–260. 12. De R, Kundu P, Swarnakar S, Ramamurthy T, Chowdhury A, Nair GB, Mukhopadhyay AK. 2009. Antimicrobial activity of curcumin against Helicobacter pylori isolates from India and during infections in mice. Antimicrob Agents Chemother 53(4):1592–1597. 13. Panchatcharam M, Miriyala S, Gayathri VS, Suguna L. 2006. Curcumin improves wound healing by modulating collagen JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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and decreasing reactive oxygen species. Mol Cell Biochem 290(1–2):87–96. Seehofer D, Schirmeier A, Bengmark S, Carter J, Koch M, ¨ Glanemann M, Nussler AK, Neuhaus P, Menger MD. 2009. Inhibitory effect of curcumin on early liver regeneration following partial hepatectomy in rats. J Surg Res 155(2):195– 200. Priya S, Sudhakaran PR. 2008. Curcumin-induced recovery from hepatic injury involves induction of apoptosis of activated hepatic stellate cells. Indian J Biochem Biophys 45(5):317–325. Lao CD, Demierre MF, Sondak VK. 2006. Targeting events in melanoma carcinogenesis for the prevention of melanoma. Expert Rev Anticancer Ther 6(11):1559–1568. Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, Ming-Shiang W, Yu HS, Jee SH, Chen GS, Chen TM, Chen CA, Lai MK, Pu YS, Pan MH, Wang YJ, Tsai CC, Hsieh CY. 2001. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or premalignant lesions. Anticancer Res 21 (4B):2895–2900. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. 1998. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med 64(4):353–356. Muller, RH, Moschwitzer FN, Bushrab FN. 2006. Manufacturing of nanoparticles by milling and homogenization techniques. In Nanoparticle technology for drug delivery; Gupta, RB, Kompella, UB, Eds. New York City, New York: Taylor & Francis Group, pp 21–52. Eerdenbrugh BV, Van den Mooter G, Augustijns P. 2008. Top-down production of drug nanocrystals: Nanosuspension stabilization, miniaturization and transformation into solid product. Int J Pharm 364:67–75.
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21. Lee J, Choi JY, Park CH. 2008. Characteristics of polymers enabling nano-comminution of water-insoluble drugs. Int J Pharm 355:328–336. 22. Choi JY, Yoo JY, Kwak HS, Nam BU, Lee J. 2005. Role of polymeric stabilizers for drug nanocrystal dispersions. Curr Appl Phys 5:472–474. ¨ 23. Muller, RH, Peters K, Becker R, Kruss B. 1995. Nanosuspensions—A novel formulation for the i.v. administration of poorly soluble drugs. Budapest, Hungary: 1st World Meeting APG/ APV, pp 491–492. 24. Peters K. 1999. Nanosuspensionen—ein neues formulierun¨ schwerl¨osliche Arzneistoffe. Ph.D. Thesis. Berlin, gsprinzip fur ¨ Berlin. Germany: Freie Universitat 25. Eerdenbrugh BV, Vermant J, Martens JA, Froyen L, Humbeeck JV, Augustijns P, Den Mooter GV. 2009. A screening study of surface stabilization during the production of drug nanocrystals. J Pharm Sci 98:2091–2103. 26. Mu L, Seow PH, Ang SN, Feng SS. 2004. Study on surfactant coating of polymeric nanoparticles for controlled delivery of anticancer drug. Colloid Polym Sci 283:58–65. 27. Janke S. 2001. The theory of high-pressure homogenization. In Dispersion techniques for laboratory and industrial scale processing; Muller RH, Bohm BHL, Eds. Stuttgart, Germany: Wissenschafttiche Verlagsgesellschaft mbH. ¨ 28. Muller, RH. 1996. Zetapotential und Partikeladung in der Laborpraxis. Stuttgart, Germany: Wissenschaftliche Verlagsgeselschaft mbH. ¨ 29. Mauludin R, Muller RH, Keck CM. 2009. Kinetic solubility and dissolution velocity of rutin nanocrystals. Eur J Pharm Sci 36:502–510. 30. R.Suryanarayanan. 1995. X-ray powder diffractometry. In Physical characterization of pharmaceutical solids; Brittain HG; Ed. New York City, New York: Marcel Dekker, pp 187–221.
DOI 10.1002/jps
Pharmaceutics Research Group, School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia
2 Freie Universit¨at Berlin, Department of Pharmacy, Pharmaceutical Technology, Biopharmaceutics & NutriCosmetics, Berlin, Germany
Received 23 June 2012; revised 25 August 2012; accepted 14 September 2012 Published online 9 October 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23335 ABSTRACT: Curcumin, a naturally occuring polyphenolic phytoconstituent, is isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae). It is water insoluble under acidic or neutral conditions but dissolves in alkaline environment. In neutral or alkaline conditions, curcumin is highly unstable undergoing rapid hydrolytic degradation to feruloyl methane and ferulic acid. Thus, the use of curcumin is limited by its poor aqueous solubility in acidic or neutral conditions and instability in alkaline pH. In the present study, curcumin nanocrystals were prepared using high-pressure homogenization, to improve its solubility. Five different stabilizers [polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), D-"-tocopherol polyethylene glycol 1000 succinate (TPGS), sodium dodecyl sulfate (SDS), carboxymethylcellulose sodium salt] possessing different stabilization mechanism were investigated. The nanoparticles were characterized with regard to size, surface charge, shape and morphology, thermal property, and crystallinity. A short-term stability study was performed storing the differently stabilized nanoparticles at 4◦ C and room temperature. PVA, PVP, TPGS, and SDS successfully produced curcumin nanoparticle with the particle size in the range of 500–700 nm. PVA, PVP, and TPGS showed similar performance in preserving the curcumin nanosuspension stability. However, PVP is the most efficient polymer to stabilize curcumin nanoparticle. This study illustrates that the developed curcumin nanoparticle held great potential as a possible approach to improve the curcumin solubility then enhancing bioavailability. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:204–214, 2013 Keywords: curcumin; nanocrystals; steric stabilization; electrostatic stabilization; polymers; high-pressure homogenization; surface charge; aggregation; crsytallinity; nanoparticle
INTRODUCTION Curcumin, a naturally occuring polyphenolic phytoconstituent, is isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae). Curcumin is chemically (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1, 6-diene-3,5,-dione. It has a pKa1 , pKa2 , and pKa3 value of 7.8, 8.5, and 9.0, respectively, for three acidic protons.1,2 It is insoluble in water under acidic or neutral conditions but dissolves in alkaline environment. Curcumin is highly unstable undergoing rapid hydrolytic degradation in neutral or alkaline conditions to feruloyl methane and ferulic acid.3 Thus, the use of curcumin is limited by its poor Correspondence to: Heni Rachmawati (Telephone: +62-222504852; Fax: +62-22-2504852; E-mail: [email protected], h [email protected]) Journal of Pharmaceutical Sciences, Vol. 102, 204–214 (2013) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
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aqueous solubility in acidic or neutral conditions and instability in alkaline pH. On pharmacological evaluation, curcumin was found to posses anticancer,4,5 antioxidant,6,7 antiinflammatory,8,9 hyperlipidemic,10,11 antibacterial,12 wound-healing,13 and hepatoprotective14,15 activities. The pharmacological efficacy of curcumin makes it a potential compound for treatment and prevention of a wide variety of human diseases. In addition, it is extremely safe upon oral administration even at very high doses, as proven in various animal models or human studies.16–18 Despite this, curcumin has not yet been approved as a therapeutic agent. The relative bioavailability of curcumin has been highlighted as a major problem for this. Curcumin is slightly absorbed in the gastrointestinal tract with the maximum solubility to be 11 ng/mL in plain aqueous buffer pH 5.0. The oral bioavailability of curcumin is very low (1% in rat). In clinical trial, quantifiable serum levels of
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curcumin were not achieved after oral administration of higher dose of curcumin (8 g/day).17 Attempts have been made to improve the solubility of curcumin by its chemical derivatization, complexation, or interaction with macromolecules, for example, gelatin, polysaccharides and proteins, and cyclodextrins. However, limited complexation, high-molecular weight of cyclodextrins, and pH of the processing medium limit their practical utility. Consequently, research still focuses on the development of formulation approaches to overcome the oral bioavailability problem. Ideal would be a universal formulation practically applicable to any drug. The drug nanocrystals are such a universal formulation approach. The special properties of drug nanocrystals are increased saturation solubility and increased surface area, both leading to an increase in the rate of dissolution and subsequently bioavailability. The nanocrystal approach can be used for all drugs for which the dissolution velocity is the rate-limiting step for absorption/bioavailibility, as for curcumin. The major production methods for drug nanocrystals are pearl milling and high-pressure homogenization (HPH),19,20 which were used here to produce curcumin nanocrystals. The stabilization ability of the stabilizers is a critical parameter determining the minimal achievable size and subsequent physical stability. The physical stability of nanocrystals is essential because aggregated nanocrystals loose the increased dissolution velocity. However, different drugs require different stabilizers. The stabilization ability of four typical sterically stabilizing polymers, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), D-"-tocopherol polyethylene glycol 1000 succinate (TPGS), and carboxymethylcellulose sodium salt (Na-CMC), were investigated. In addition, a lowmolecular weight surfactant, sodium dodecyl sulfate (SDS) was used as comparison for an electrostatic stabilizer. The obtained nanocrystals were characterized in term of size and particle charge [zeta potential (ZP)]. To examine the physical stability, a short-term stability study was performed storing the differently stabilized nanosuspensions at two different temperatures.
MATERIALS AND METHODS Materials Curcumin extract from Curcuma xanthorrhiza rhizome (purity 95%) was supplied by PT Phytochemindo Reksa (Bogor, Indonesia). The stabilizers PVA (MW = 31 K; Fluka Chemica GmbH, Steinheim, Germany), PVP (MW = 25 Kda; Sigma–Aldrich Chemie GmbH, Steinheim, Germany), TPGS (MW = 1513 da; Eastman Chemical Company, Liverpool, UK), Na-CMC (MW = 90 Kda; Sigma–Aldrich Chemie), and SDS DOI 10.1002/jps
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(MW = 288 da; Fluka Chemie GmbH) were used in this study. Ultra purified water (milli-Q; Millipore GmbH, Darmstadt, Germany) was used for all formulations. The water conductivity used for ZP analysis is 50 :S/cm. The other chemicals were of analytical reagent grade. Methods
Preparation of Nanosuspensions Nanosuspensions with different stabilizers were produced by HPH using a Micron Lab 40 (APV Deutchland GmbH, Unna D-59425, Germany) with a batch size of 40 mL. The best formulation of nanosuspensions for homogenization process contained 5.0% (w/ w) curcumin and 1.0% (w/w) of each stabilizer, except for SDS, which was 0.2% (w/w). Before the homogenization, the dispersion medium was prepared by dissolving the stabilizer in water. Curcumin powder was poured into the aqueous stabilizer solution under magnetic stirring (IKA-combimag RCT; IKALabortechnik, Staufen 79219, Germany) for 2 min. After dispersion, the suspension was then transferred into the bulk container of the homogenizer and homogenization was run with two cycles at 300, 500, and subsequently 1000 bar, as premilling steps. Afterward, HPH at 1500 bar was conducted applying 20 cycles. The progress of homogenization was controlled by size analysis of samples taken after 1, 5, 10, 15, and 20 homogenization cycles.
Particle Size Analyses Photon correlation spectroscopy (PCS; Zetasizer Nano ZS; Malvern Instruments, Malvern, UK) and laser diffractometry (LD; Coulter LS 230; BeckmanCoulter, Krefeld 47807, Germany) were employed to determine the particle size of curcumin nanosuspensions. PCS yields the mean diameter of the bulk population (z-average) and the polydispersity index (PI) as measure for the width of the size distribution. The measuring range of the zetasizer is approximately 0.6 nm to 6 :m. Larger particles in the micrometer range were therefore measured with LD yielding volume-weighted diameters. Analysis of the diffraction patterns was performed using the Mie theory, with a dispersant refractive index of 1.33, a particle refractive index with a real part of 1.50, and an imaginary part of 0.01. The results are given as diameter d(v)50%, d(v)95%, and d(v)99% values, representing the percentage of particles below given size (:m).
ZP Determination The ZP of the nanosuspensions was analyzed directly after production using the Zetasizer Nano ZS (Malvern Instruments). For this purpose, the zetasizer was switched from PCS mode to the laser doppler anemometry mode. The measurement is a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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particle electrophoresis, the particle velocity is determined via the Doppler shift of the laser light scattered by the moving particles. The field strength applied was 20 V/cm. The electrophoretic mobility was converted to the ZP in mV using the Helmholtz– Smoluchowski equation. To study different aspects of the adsorption of ionic and nonionic stabilizers, different measuring media were applied (distilled water and original dispersion media). Bidistilled water having its conductivity adjusted to 50 :S/cm with 0.9% NaCl solution was used to determine the Stern potential being directly correlated to the surface charge of the nanocrystals (Nernst potential). Using a standard conductivity adjusted with 1:1 electrolyte avoids fluctuation in the ZP because of the variations in the conductivity of distilled water especially when the sample itself contains electrolytes when adding it to the distilled water. In addition, the ZP was also measured in the original media of the nanosuspension as a measure of the thickness of the diffuse layer. The higher the measured ZP in these conditions, the thicker the diffuse layer and the more stable is the suspension. On the basis of the ZP data, long-term stability can be estimated.
Microscopic Analysis A Leitz-Orthoplan (Wetzlar, Deutchland) microscope was used. Samples were analyzed with polarized light at 160-fold magnification to obtain an overview information of the whole system and for the detection of crystals or aggregates >1 :m.
Nanocurcumin Morphology The morphology of PVP-nanocurcumin was observed using scanning electron microscope (SEM). SEM was performed on nanocurcumin produced using PVP as a stabilizer, which is appointed as the most appropriate polymer for this study. The samples were fixed on a brass stub using double-sided tape and then gold coated in vacuum by a sputter coater. The pictures were taken at excitation voltage of 10 kV and at 10,000–40,000× magnification by using JSM-6360LA Scanning Microscope (Jeol, Tokyo, Japan).
Thermal Property of Nanocurcumin The thermal characteristic of curcumin and PVPnanocurcumin was analyzed using differential thermal calorimetry (DSC). The DSC thermogram of curcumin and PVP-nanocurcumin were recorded using a differential scanning calorimeter (NETZSCH STA 449F1 STA449F1A-0164-M). Approximately 10 mg of each sample was heated in an open aluminum pan from 20◦ C to 300◦ C at scanning rate of 10◦ C/min under a stream of nitrogen. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
Crystallinity of Nanocurcumin The crystalline phase of both curcumin and PVPnanocurcumin were determined by X-ray powder diffraction (XPRD) by using a Diano (Woburn, Massachusetts) diffractometer with Cu-K" radiation (λ = 0.1540 nm). A scan rate of 0.04◦ /s was applied to record the pattern in the 2θ range of 20◦ –70◦ .
Stability Test of the Nanosuspension To investigate the effect of the stabilizers on the physical stability of the nanosuspensions, a short-term stability test was performed. The nanosuspensions were stored in sealed vials at different temperatures [room temperature (RT) and 4◦ C] for 30 days. Samples were taken on day 0 (day of production), day 7, and day 30. Characterization was carried out regarding particle size analyzed by PCS, LD, and polarized light microscopy.
RESULTS AND DISCUSSION Particle Size and Size Distribution as a Function of Homogenization Cycles and Type of the Stabilizer Nanoparticles are inevitably more unstable than microparticles because of the extra Gibbs free energy contribution related to the particle size and primarily due to the surface energy.21 A careful evaluation of the type and concentration of the stabilizer used is the key to the production of physically stable nanosuspensions. Five formulations of nanosuspension were prepared in this study with 1% (w/w) of PVA, PVP, Vitamin E TPGS, Na-CMC, and 0.2% (w/w) of SDS as stabilizers, respectively. In case of polymers used in the preparation of nanocrystals, these molecules should adsorb onto the surfaces of drug nanocrystals and provide steric stabilization effect.22 When their concentration is very low, it is often the case that the polymers bridge the particles and the dispersion is destabilized. According to our experience, the dispersion is usually stabilized by increasing the polymer concentration to the order of 1%. This is because most of the surfaces of the particles are covered by the adsorbed polymers. The adsorbed chain molecules on surface have ceaseless thermal motion, resulting in dynamically rough surface preventing coalescence by repulsive entropic force. Preliminary study was performed to optimize the concentration of each stabilizer, based on the process feasibility. The curcumin crystalline powder as received had LD d(v)50% of 5.89 :m and d(v)99% of 105.99 :m. The influence of the homogenization cycles on the particle size and size distribution is shown in Figure 1. After 20 cycles, smallest size was obtained for the TPGS stabilized system, the size increased in the order of: TPGS < SDS < PVP < PVA < Na-CMC. DOI 10.1002/jps
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Figure 1. Particle size (PCS, z-average) and width of the size distribution (polydispersity index, PI) as function of homogenization cycles for the five different curcumin nanosuspensions. (PM = values after premilling.)
It was described that the resulting final nanocrystal size does not depend on the type of stabilizer.23,24 However, different sizes were obtained by different types of stabilizers. As expected, SDS as well dispersing, fast diffusing low-molecular weight surfactant should reveal the best performance in the nanoparticle production. SDS-stabilized suspension effectively produced nanoparticle after five homogenization cycles with average size of particle about 630 nm and a PI of 0.56. Further homogenization cycles only changed a minor size reduction with steady in the particle distribution. However, TPGS exerted even better effectivity in producing crystals being in the nanometer range with narrow distribution just after premilling. Further homogenization cycles, that is, after 10 cycles, only slightly decreased the particle size and the steady state value was achieved with the size of about 550 nm and a PI of 0.47. This result can be explained by considering the chemical structure of TPGS. TPGS comprises amphiphilic properties, with a hydrophilic–lipophilic balance of about DOI 10.1002/jps
13. Moreover, its hydrophilic tail (polyethylene glycol) and hydrophobic portion (tocopherol succinate) are bulky and have large surface areas. Such characteristic makes it a good stabilizer. The performance of TPGS can be explained by the combination of low viscosity and high surface activity.25,26 This is a characteristic of advantages, which makes TPGS superior to other stabilizers used in this production. The ability to quickly cover depends on the molecular weight of the surfactants (diffusion velocity to the particle surface). The lower the molecular weight of surfactants, the faster they diffuse and vice versa. This was confirmed in this study. The higher the molecular weight of polymers (in case of PVA and Na-CMC), the slower was the decrease in particle size with increasing number of cycles (Fig. 1). For that reason, PVP with lower molecular weight than PVA and Na-CMC exhibits faster size reduction and stabilization. Na-CMC showed the least efficient size reduction. Smallest size (824 nm) was reached after 15 cycles. At 20 cycles, the size increased to 878 nm. This can be explained that a too JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 2. Particle diameter d(v)50%, d(v)95%, and d(v)99% (LD data) as a function of homogenization cycles for the five different curcumin nanosuspensions.
high input of energy in the homogenization process leads to aggregation in suspensions.27 The larger PCS sizes of the polymer-stabilized nanocrystals can be explained by aggregation due to the polymer bridging. The steady decrease in PI as shown in Figure 1 for all nanosuspensions confirms that at higher cycles mainly remaining larger particles are removed, and only limited further size reduction of the bulk population takes place. The influence of different stabilizers in producing curcumin nanosuspension can also be seen from LD data (Fig. 2). There is no change in the diameter d(v)50% characterizing the bulk population when moving from cycle 10 to 20, for all nanosuspensions. Only Na-CMC-stabilized nanosuspension showed significant reduction in d(v)95% when the homogenization cycle was increased from 15 to 20. A further more or less pronounced decrease was observed for all nanosuspensions in the diameter d(v)99%, being a sensitive measure of the presence of even a few larger particles. According to the LD data, again TPGS exerted the best stabilizing performance, whereas Na-CMC is the least efficient. The main reason for this different ability in the formation and stabilization of generated nanocrystals appears to be the stabilizer nature. The choice of stabilizer is therefore specific to each drug candidate and each formulation procedure. To preserve the nanocrystals from aggregation, the stabilizer should exhibit sufficient affinity on the particle surface. When Na-CMC with molar mass of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
90 Kda was used, the particle diameter of the resulting dispersion is larger. This means the addition of an anionic polyelectrolyte of high molar mass increases the tendency to form larger particle aggregates, attributed to polymer bridging. It should provide better stabilization than PVP or PVA because in Na-CMC steric and electrical stabilization principles are combined. ZP Determination The values of ZP of five differently stabilized nanosuspensions are presented in Table 1. In regard to the successful nanocrystal production reflected in the PCS and LD profiles above (Figs. 1 and 2), there is a close relationship between the ZP value and the size characteristic of obtained nanocrystals. SDSstabilized nanosuspension has the highest ZP value Table 1. Zeta Potential Values of the Five Differently Stabilized Curcumin Nanosuspensions; Left: Analyzed in Water (Conductivity 50 :S/cm, pH 5.8); Right: Analyzed in the Original Dispersion Medium
Stabilizer
Analyzed in Water (mV)
Analyzed in Original Dispersion Medium (mV)
Conductivity (:S/cm)
PVA PVP TPGS SDS Na-CMC
−5.5 −15.9 −12.5 −30.6 −26.6
−2.3 −11.9 −15.9 −52.2 −37.7
131 40 9 2640 1849
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Figure 3. Scanning electron microscopic photograph of curcumin (a) and PVP-nanocurcumin (b). Magnification 40,000×.
(−30.6 in water and −52.2 in SDS solution) among others even at low concentration (0.2%, w/w). SDS, an excellent electrostatic stabilizer, adsorbs the negatively charged alkyl chain of the molecule with high affinity onto particle surfaces leading to a high ZP. This high ZP of SDS is in accordance to the PCS diameter, in which after five cycles of homogenization, the most pronounced decrease took place. Both particle size and distribution remained unchanged up to cycle 20. This confirms that aggregate formation during the homogenization process—input of more energy is applied—can be avoided by the presence of SDS on the surface. The higher ZP of SDS-stabilized nanosuspension measured in original dispersion medium (SDS solution) is in agreement with the ZP theory. The negatively charged part of the molecule (alkyl chain with SO4 2− ) was adsorbed onto the particle surface and leading to an inner Helmholtz adsorption layer more densely packed than in distilled water. Adsorption takes place according to the theory of adsorption isotherms.28 Consequently, the potential of the inner Helmholtz layer increases leading subsequently to an increase of the ZP. As an ionic high-molecular weight polymer, Na-CMC provides a combination of electrostatic and steric stabilizations. An increased adsorption can also explain the increase of the ZP value of Na-CMC-stabilized nanosuspension when measured in the Na-CMC solution. The increase of the ZP in the original dispersion medium is similar high as the observed increase in SDS-stabilized nanosuspension (approximately by 20 mV). The increase of the potential of the inner Helmholtz layer due to the increased adsorption of negatively charged carboxylic (COO− ) groups onto particle surface has obviously more predominant effect than the shift of plain of shear due to the adsorption layer of the polymer chain. In addition, the negative charges of the Na-CMC are in but also on the surface of the polymer layer, thus increasing the Stern and subsequently ZP. The net result is therefore an increase in ZP from −26 to −37 mV. The ZP is higher than 30 mV, that is, above the limit required DOI 10.1002/jps
for physically stable dispersion systems.28 The ZP is therefore not the reason why nanosuspension production using Na-CMC was less effective compared with the SDS (Figs. 1 and 2). This supports the assumption that the aggregate formation was caused by bridging. In contrast, nanosuspensions with steric stabilizers (PVA, PVP, and TPGS) exhibited lower ZP values in water (−5.5, −15.9, and −12.5 mV, respectively). The adsorption layer of these polymers shift the plain of shear, at which the ZP is measured, to a larger distance from the particle surface. Consequently, the measured ZP is lower. The measured ZP values for the three nonionic steric stabilizers are similar in original dispersion medium and in distilled water. This indicates no desorption when the nanosuspensions are diluted with water for the measurement. This can be explained by the multipoint attachment of adsorbed polymers. In contrast the low-molecular weight, end-on (= single point) attached SDS is desorbed, the ZP decreases from about −52 to −31 mV. Of course, Na-CMC is also a polymer with ability to multipoint attachment. However, binding to the surface is less firm compared with the nonionic polymers. The repulsion between the negatively charged nanocrystal surface and the negatively charged CMC chains weakens the adsorptive forces, leading to partial desorption upon dilution. The measured ZP decreases. However, it does not indicate that these nanosuspensions exhibiting low physical stability as the stabilization occur through a steric mechanism. Thus, a reduction of the measured ZP is not necessarily correlated with a reduced stability. As the particle charge is one of the factors determining the physical stability of dispersion system, the ZP value can be used to predict the physical stability of these suspensions. Considering the obtained ZP results, it could be judged that all the investigated stabilizers should be suitable for the stabilization of curcumin nanosuspension. However, Na-CMC was not being a nice example for the destabilizing effect of polymer interactions. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 4. DSC of curcumin (a) and PVP-nanocurcumin (b).
Morphology of PVP-Nanocurcumin
Thermal Characteristic of PVP-Nanocurcumin
Figure 3 shows the morphologies of curcumin and PVP-nanocurcumin with aggregated status. Spherical morphology with a highly porous, foamlike structure can be observed only in curcumin nanoform. Particle size of the nanoparticles was drastically decreased from microscale to nanoscale (±400 nm).
Differential thermal calorimetry detects phase transition: glass transition (exothermic), crystallization, and (endothermic) melting. DSC thermograms were obtained to define the physical state of the drug and polymer in the nanoparticles and to detect any drug polymer interactions within the PVPnanocurcumin. Typical thermograms of curcumin and
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Figure 5. XPRD of curcumin (red line) and PVP-nanocurcumin (blue line).
PVP-nanocurcumin were shown in Figure 4. The purpose of these analyses was to detect possible alteration in the structure and nature of compound due to their organization in the form of nanoparticle. The DSC curve of curcumin as well as its nanoform showed a single endothermic peak at 180.5◦ C and 173.9◦ C, respectively. These results suggest that the homogenization process did not change the crystallinity of curcumin. The slightly changes in the melting temperature may be due to the presence of PVP in the mixture as also confirmed by other study.29 Crystallinity of PVP-Nanocurcumin Crystallinity characteristic are analyzed with XPRD when diffraction pattern of the X-ray from the sample is determined as a function of scattering angle.30 XPRD analysis of curcumin and PVP-nanocurcumin were performed and are illustrated (Fig. 5). No other peaks were observed, which is indicating the high purity of the samples. The XPRD data clearly explained that after homogenization, the nanocurcumin remains in the crystalline state although with less intensity. This seems to be in line with the DSC data, which suggests any slightly alteration in the crystallinity. The low percentage of PVP in the formulation (1%) seems not significantly interfering the diffractogram of PVP-nanocurcumin. Short-Term Stability Test of Curcumin Nanosuspension Although the number of articles describing nanosuspension formulations is extensive, limited literature DOI 10.1002/jps
is currently available that aims to evaluate and compare the ability of different stabilizers in their stabilizing potential from a more fundamental point of view, as reported here. The manufacturing of a nanosuspension implies the creation of additional surface area and hence interface. The nanosuspensions formed are thermodynamically less stable and tend to minimize their total energy by agglomeration. Kinetically, the process of agglomeration depends on its activation energy, which can be influenced by the presence of stabilizers to the system.20 To evaluate the stabilizing potential of stabilizers during storage, a stability test for 30 days was performed at two different temperatures (25◦ C and 4◦ C). The parameters observed in this study are particle size (Fig. 6) and polarized light microscopy (Fig. 7), performed after the nanosuspensions were stored for 7 and 30 days. The effect of different stabilizers on the physical stability of nanosuspensions is reflected by the PCS mean diameter and the LD diameter d(v)95% and d(v)99% (Fig. 6). The sterically stabilized nanosuspensions (PVA, PVP, and TPGS) exhibited a good performance in preventing particles from agglomeration at both temperatures. No or little changes in LD and PCS diameters were detected, except for PVA-stabilized nanosuspension minor, negligible increased in PCS diameter was observed. In accordance with the PCS and LD data, the polarized micrograph revealed no detectable crystal growth or agglomeration (Fig. 7). To summarize, the nanosuspensions JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
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Figure 6. PCS diameter and LD diameter d(v)95% and d(v)99% of five differently stabilized curcumin nanosuspensions as function of days (0–30) stored at room temperature (RT, left) and at 4◦ C (right).
stabilized with PVA, PVP, and TPGS are stable over period of 30 days at both storage conditions. On the basis of the ZP data and the small sized obtained, SDS-stabilized nanosuspension was expected to be physically stable. However, even a marked increase both of d(v)95% and d(v)99% was observed. The increase in LD diameter indicates agglomeration during the storage. The formed agglomerates were not dispersed by the instrument stirring in the sample cell during LD measurement. This indicates strong interactive forces in the agglomerates because loose agglomerates are dispersed by dispersing forces in the sample cell. Moreover, polarized light micrograph (Fig. 7) clearly proved that after 7 and 30 days of storage, agglomerations took place. The decrease in PCS size can be explained that small sized aggregates (a few :m) continued to agglomerate increasing in size leaving the measuring range of PCS. The small sized, more stable, and nonaggregated nanocrystals remained. It is indeed contradictive with the ZP value, which is actually in the range JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 1, JANUARY 2013
of good stability (>30 mV). The potential reason to explain this situation is that SDS formed a double layer on the particle surface, which hydrophobized the surface. This promotes hydrophobic interaction leading to particle aggregation. In contrast with the nanosuspension stabilized by Na-CMC, a significant increase in PCS diameter in time was observed, especially when stored at RT. However, no change in LD size was detected. The relatively constant diameter observed in LD measurements due to the formation of loose aggregates. They were easily deaggregated by stirring during the measurements as observed previously for hesperetin nanosuspensions [not published yet]. This argument is confirmed by the polarized micrograph (Fig. 7), which clearly showed agglomerate growth over 7 and 30 days of storage. PCS measurements showed an increase in the PCS diameter, attributed to slow continuing agglomeration of nanocrystals in the measuring range of PCS. Deagglomeration did not take place during the measurement because the samples are not DOI 10.1002/jps
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lize the produced crystals in the concentration used. PVA, PVP, and TPGS showed similar performance in preserving the curcumin nanosuspension stability. However, for further pharmaceutical processing reason, PVP is suggested as the most efficient polymer to stabilize curcumin nanocrystal. Oral curcumin nanocrystals appear as a potential natural product in the forefront of therapeutic agents for treatment of human diseases.
ACKNOWLEDGMENTS This project was financially supported by DAAD (German Academic Exchange Service), under scheme of Indonesian–German Scientists Exchange Program.
REFERENCES
Figure 7. Polarized light micrographs (magnification 160×) of the nanosuspensions after 7 and 30 days of storage at RT (7D and 30D). Nanosuspension stabilized with nonionic polymers (PVA, PVP, and TPGS) are stable with no agglomeration detected, in contrast to ionic stabilized nanosuspensions (SDS and Na-CMC).
stirred. Considering these results, it can be concluded that Na-CMC is not sufficient to stabilize aqueous curcumin nanosuspensions.
CONCLUSIONS Nanocrystals of curcumin were succesfully produced with four stabilizers PVA, PVP, TPGS, and SDS with the particle size in the range of 500–700 nm. Na-CMC resulted in an only slightly larger PCS diameter (about 800 nm) but distinctly higher diameter d(v)99%, indicating presence of aggregates. This indicates that this polymer is not able to efficiently stabiDOI 10.1002/jps
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DOI 10.1002/jps