Curcumin

Accepted Manuscript Curcumin-Eudragit® E PO solid dispersion: a simple and potent method to solve the problems of curcumin Jinglei Li, Il Woo Lee, Gye Hwa Shin, Xiguang Chen, Hyun Jin Park PII: DOI: Reference:

S0939-6411(15)00261-1 http://dx.doi.org/10.1016/j.ejpb.2015.06.002 EJPB 11959

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

12 March 2015 14 May 2015 5 June 2015

Please cite this article as: J. Li, I.W. Lee, G.H. Shin, X. Chen, H.J. Park, Curcumin-Eudragit® E PO solid dispersion: a simple and potent method to solve the problems of curcumin, European Journal of Pharmaceutics and Biopharmaceutics (2015), doi: http://dx.doi.org/10.1016/j.ejpb.2015.06.002

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Curcumin-Eudragit® E PO solid dispersion: a simple and potent method to solve the problems of curcumin Jinglei Li,a Il Woo Lee,a Gye Hwa Shin,a Xiguang Chen,b Hyun Jin Parka* a

School of Life Sciences and Biotechnology, Korea University, 5-Ka, Anam-Dong, Sungbuk-Ku,

Seoul 136-701, Republic of Korea b

College of Marine Life Science, Ocean University of China, Qingdao, 266003, Shandong, PR

China

* To whom correspondence should be addressed: Hyun Jin Park, Professor, School of Life Sciences and Biotechnology, Korea University, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea;

Tel.: 82-2-3290-3450; Fax: 82-2-953-5892;

E-mail address: [email protected]

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Abstract

Using a simple solution mixing method, curcumin was dispersed in the matrix of Eudragit® E PO polymer. Water solubility of curcumin in curcumin-Eudragit® E PO solid dispersion (Cur@EPO) was greatly increased. Based on the results of several tests, curcumin was demonstrated to exist in the polymer matrix in amorphous state. The interaction between curcumin and the polymer was investigated through Fourier transform infrared spectroscopy and 1

H NMR which implied that OH group of curcumin and carbonyl group of the polymer involved

in the H bonding formation. Cur@EPO also provided protection function for curcumin as verified by the pH challenge and UV irradiation test. The pH value influenced curcumin release profile in which sustained release pattern was revealed. Additionally, in vitro transdermal test was conducted to assess the potential of Cur@EPO as a vehicle to delivery curcumin through this alternative administration route.

Keywords: Curcumin, Eudragit® E PO, solid dispersion, water solubility, stability, transdermal administration

Chemical compounds studied in this article:

Curcumin (PubChem CID: 969516); Eudragit® E PO (PubChem CID: 107676); Sodium Acetate (PubChem CID: 517045); Acetone (PubChem CID: 180)

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1. Introduction

Curcumin [(E,E)-1,7-bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-ione] is the primary active ingredient of the perennial herb Curcuma Longa (turmeric) which has been used as nutritional supplement and herbal medicine in many Asian countries for thousands of years [1]. In recent decades, curcumin has been extensively investigated for its wide spectrum of therapeutic properties, such as antioxidant, anti-inflammatory, anti-cancer, antimicrobial, wound healing, and potential prevention ability of neurodegenerative diseases [2-5]. Not like other phytochemicals and most synthetic chemotherapeutic agents, curcumin can affect more than one hundred molecular targets and many different pathways which endows curcumin particular edge over its counterparts [6]. Examined by thousands of years’ usage and several clinical trials, curcumin has very good safety profile: as high as 8 g/day dosage cause no adverse effects [7].

However, the optimistic estimation of curcumin as a magical panacea faces several pitfalls associated with this interesting polyphenolic compound. Curcumin has extremely low solubility and dissolution rate in aqueous media due to strong inter and intra-molecular hydrogen bonds [6]. It has slightly higher solubility in alkaline solution but is degraded quickly into vanillin, ferulic acid, and feruloyl methane [8]. Environmental factors like UV irradiation also quickly decompose curcumin, both in the form of crystalline and solution [9]. Moreover, curcumin has very low bioavailability as revealed in many animal and human trails [10]. After absorption in the gastrointestinal track, curcumin undergoes quick decomposition through first pass

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metabolism which greatly reduce the effective concentration of curcumin in blood plasma [10, 11].

Several methods have been proposed and investigated to circumvent the inherent limitations of curcumin. One approach involves encapsulation of curcumin into various nano-carriers such as nano-liposome, nano-emulsion, nano-lipid particle and micelles [12]. Using these methods, the water solubility can be increased by several hundred folds [13]. But most of the methods reported offer curcumin little protection under UV and higher pH challenge [14]. And most of the preparation methods are very complicated and require large amount of organic solvent which is also problematic. Other methods attempt to modify curcumin chemical reaction by chemical reaction to increase its water solubility and stability [15]. However, apart from the rigorous reaction conditions, the modified curcumin tends to loss its functionalities, at least to some degree.

Recently, solid dispersion method was also reported to overcome the problems associated with curcumin [16, 17]. Not like nano and chemical modification methods, solid dispersion is relatively easy to prepare, with higher loading ability and stability. It is also scalable and economic which makes this method particular appealing in the pharmaceutical industry. On the other hand, transdermal administration can bypass the first pass metabolism and proved to be a promising alternative to the conventional administration routes [18]. Nano-emulsion, liposome,

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hydrogel and many other composites have been reported to delivery curcumin through transdermal route [19-22].

Eudragit® E PO is a cationic polyelectrolyte that belongs to the family of (meth) acrylate copolymers. It is composed of dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate with the molar ratio of 2:1:1 [23]. It is widely used in the pharmaceutical industry for various purposes such as taste masking, moisture protection, release modification and excipient [23]. Since Eudragit® E PO bears positive charge, when dissolved in aqueous media it can be utilized to prepare solid dispersion with negatively charged drug by various methods [24, 25].

In the present study, we developed curcumin-Eudragit® E PO solid dispersion (Cur@EPO) through solution mixing method. Cur@EPO powder shows the characteristic color of curcumin and can be dissolved easily in acidic aqueous solution. The prepared solid dispersion not only increased the water solubility but also greatly increased its stability against higher pH and UV irradiation. We investigated the possible mechanism that responsible for the observed phenomena via DSC, XRD, FT-IR and 1H NMR measurements. At last, we examined the in vitro transdermal release of curcumin using a vertical modified amber glass Franz diffusion cell. The results of this study indicate that curcumin-Eudragit® E PO solid dispersion is a very promising mean to circumvent the problems of curcumin and to extend its potential pharmacological applications.

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2. Materials and methods

2.1 Materials

Curcumin with 98% purity was purchased from Acros Orangnics (New Jersey, USA). Eudragit® E PO with molecular weight about 150 kDa was provided by Evonik Industries AG (Darmstadt, Germany) and was used as received. Membrane tubing (MWCO: 2000) was obtained from Spectrum Laboratories (Rancho Dominquez, California, USA). Strat-M membrane was provided by Merck KGaA (Darmstadt, Germany). All other reagents and solvents were of analytical grade and used as received.

2.2 Preparation procedure

The preparation method for Cur@EPO was illustrated in Scheme 1. Frist of all, Eudragit® E PO was dissolved in sodium acetate buffer solution, pH 4.5, at the concentration of 1 mg/mL. Then, certain volume of curcumin acetone solution (about 4 mg/mL) was dropped into Eudragit® E PO solution under magnetic stirring at the speed of 1500 rpm. Acetone was removed by rotary evaporation. Cur@EPO powder was retrieved after 48 h of lyophilization at -60℃.

2.3 Loading ability and efficiency

Loading ability was defined as the weight ratio of curcumin to Eudragit® E PO while loading efficiency was defined as ratio of loaded curcumin to initial amount of curcumin. The content of curcumin in Cur@EPO was calculated according to a calibration formula listed below:

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Y=0.1445X-0.0152, r2=0.9999;

where Y was the absorption at 419 nm and X was the concentration of curcumin.

The calibration curve was obtained from the absorption readings at 419 nm of several standard curcumin ethanoic solutions which were prepared by dissolving curcumin in ethanol diluted by sodium acetate buffer solution (pH 4.5) (9:1 v/v). Cur@EPO dissolved in sodium acetate buffer solution was mixed with 9 times of ethanol and the absorption at 419 nm was recorded. All the tests were done in triplicate and results were expressed as means and standard deviation.

2.4 Hydrodynamic size and zeta potential analysis

Size distribution was measured by dynamic light scattering (DLS) on a nano-ZS nanosize analyzer (Malvern, Worcestershire, U.K.). About 1 mL of sample solution was transferred to a zeta cell, and the measurement was performed at 25℃ with a detector angle of 90° and wavelength of 633 nm. Zeta potential was also recorded on the same equipment according to our previous study [12].

2.5 Transmission Electron Microscopy (TEM) analysis

In our pilot study, we found that curcumin nanoparticle was formed when curcumin organic solution was dropped into distilled water without any surfactant or stabilizer. But the curcumin nanoparticle was unstable and quickly precipitated out of the solution within 24 h. This is in agreement with earlier reports [26, 27]. The morphology of curcumin nanoparticle and

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Cur@EPO was observed by TEM analysis (Philips-FEI, Eindhoven, Netherlands). One drop of the freshly prepared curcumin nanoparticle solution or Cur@EPO sodium acetate buffer solution (1 mg/mL) was deposited onto the carbon-coated grids and kept for 1 min. Excess solution was removed by filter paper. Then one drop of phosphotungstic acid solution (2% in distilled water) was applied to stain the sample for about 1 min as reported previously [13]. All the samples were dried in room temperature overnight (12 h) before capturing the TEM images.

2.6 Field emission scanning electron microscopy (FE-SEM) analysis

The morphology of freeze dried powder of Cur@EPO was observed via an S-4300 field emission scanning electron microscopy (FE-SEM, Hitachi, Tokyo, Japan) at an accelerating voltage of 15 kV. All the samples were fixed on a metallic stub surface by double-sided carbon tape, and then coated with platinum in an ion sputter under vacuum for 90 s before the observation.

2.7 Differential scanning calorimetry (DSC)

Thermal transition properties of curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, and Cur@EPO were analyzed by a differential scanning calorimeter (Seiko DSC 6100, Chiba, Japan). Physical mixture was prepared by mixing curcumin and Eudragit® E PO powder at the weight ratio of 20:80 with a mortar and pestle. Sample powder (about 1.50 mg) was weighted and sealed in an aluminum DSC pan. Scanning was performed from room

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temperature to 300℃ at a heating rate of 10℃/min under dry nitrogen purge of 50 mL/min. An empty pan was served as reference.

2.8 X-Ray diffraction (XRD)

Crystalline phase was identified with XRD analysis. The measurement was conducted on an X-ray diffractometer (X’Pert PW3040/00, Philips, Almelo, Netherlands) equipped with Cu-Kα-radiation with K-A2/K-A1 ratio of 0.5, generated at 30 mA and 40 kV. The scanning was operated in the range between 5-40° 2θ with a step angle of 0.02° at a scan rate of 1°/min.

2.9 Fourier-transform infrared (FT-IR) investigation

The possible interaction between curcumin and Eudragit® E PO was investigated by FT-IR. All the samples (curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, Cur@EPO) were prepared using the KBr pallet method as reported in the previous publication [28]. Spectra of samples were recorded on a Varian 640-IR (California, USA) in the wavelength region between 600 and 4000 cm−1. Each spectrum was obtained by averaging 32 scans at a resolution of 4 cm-1. 2.10 1H NMR analysis Solution 1H NMR spectra of curcumin, Eudragit® E PO and Cur@EPO was recorded on a Varian Unity Plus 600 spectrometer at 25℃. Curcumin was dissolved in acetonitrile-d3 and all

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other samples were dissolved in a mixture solution of acetic acid-d4 with D2O (2:98 v/v). Sample solution was transferred to a 5 mm diameter tube before the measurement.

2.11 Stability test

Curcumin is unstable in alkaline environment and under UV irradiation. Nevertheless, the stability of drug in aforementioned conditions is highly desirable in pharmaceutical industry. Herein, we tested the stability of Cur@EPO under pH challenge and UV irradiation test. All the tests were done in triplicate and results were expressed as means and standard deviation.

2.11.1 pH stability test

Freeze dried Cur@EPO samples and raw curcumin were dissolved in sodium acetate buffer solution (pH 4.5) and ethanol respectively. Small portion (0.1 mL) of the solution was mixed with 10 mL of PBS buffer at different pH values. The absorptions at 419 nm were recorded immediately and the readings were regarded as 100% curcumin remaining. Absorption was measured at predetermined time points within 24 h. To exclude the possible effect of sodium acetate buffer solution to the pH of PBS, the same volume of sodium acetate buffer solution was added into the PBS that used to dissolve curcumin. And during the test, all the samples were kept in a dark box.

2.11.2 Direct UV photolysis

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In order to determine UV photolysis protection function of Cur@EPO for curcumin, a direct photolysis experiment was carried out using the freeze dried form of Cur@EPO. A UV lamp (G4T5E UV-B, Sankyo Denki, Tokyo, Japan) was applied to mimic the UV rays of sunlight (290–360 nm). About 2 mg of Cur@EPO powder was weighed to a small glass vail. For curcumin, 0.2 mL of curcumin acetone solution (1.5 mg/mL) was added to a glass vail and the solvent was evaporated under reduced pressure. The prepared samples were irradiated under the UV illumination at room temperature in a dark box for 24 h. The distance between the bottom of the vials and the UV lamp was 10 cm. After irradiation for a predetermined time duration, Cur@EPO samples were dissolved in sodium acetate buffer solution while curcumin was dissolved in ethanol for determining the absorption at 419 nm on a UV-vis spectrophotometer. The absorptions of sample solutions at 419 nm without UV treatment were recorded and regarded as 100% curcumin remaining.

2.11.3 Indirect UV photolysis

Indirect photolysis was performed in solution to determine the photoprotection capacity of Cur@EPO to curcumin. Curcumin was dissolved in ethanol at the concentration of 20 ppm while Cur@EPO was dissolved in sodium acetate buffer solution at the concentration of 1 mg/mL. Indirect photolysis conditions were similar to that of direct photolysis. At predetermined time point, the absorption at 419 nm was recorded. Absorptions of sample solutions without UV treatment were measured and regarded as 100% curcumin remaining.

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2.12 Drug release profile

Curcumin release profile at different pH values was investigated to examine the impact of pH on the release of curcumin from Eudragit® E PO via a modified dialysis method [29]. After dissolving Cur@EPO 16% in sodium acetate buffer solution at the concentration of 1 mg/mL, 1 mL solution was pipetted into a small glass vail which was tightly sealed with unilaminar dialysis tubing membrane (MWCO: 2000) by a rubber band. The glass vail was inverted into a larger glass vail filled with 50 mL of release medium (PBS, pH 4.5, 6.5 and 7.4, or 0.1 N HCl solution) which contained Tween 80 (1 wt%) under stirring. At predetermined time intervals, 1 mL release medium was withdrawn for analyzing the concentration of curcumin on a UV spectrophotometer. Fresh release medium was added immediately to keep the sink condition. The release was monitored within 48 h.

2.13 In vitro transdermal test

In vitro skin penetration test was performed on a vertical modified amber glass Franz diffusion cell (Daihan Labtech, Gyeonggi-do, South Korea) according to our previous report [30]. A synthetic membrane (Strat-M) was used instead of animal or human skin. The synthetic membrane can be used to mimic the characteristics of human skin and avoid certain ethic issues. After filled the receiver compartment with 10 mL of PBS, pH 7.4, containing Tween 80 (1 wt%), a Strat-M membrane was sandwiched and fastened between the donor and receiver compartments. The system was equilibrated at 37 ± 1 °C for 1 h before 0.5 mL of sample

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solution (Cur@EPO 16% sodium acetate buffer solution, curcumin DMSO solution and fresh curcumin nanoparticle solution) was dropped slowly on the membrane to cover the whole membrane surface. The solution in receptor was continuously stirred at 600 rpm during the period of test. At predetermined time point, 1 mL of receptor medium was withdrawn and analyzed for the concentration of curcumin. To fill the withdrawn amount, 1 mL of fresh medium was carefully added to the receptor compartment.

3. Results and discussion

Curcumin may be one of the most studied phytotherapeutics thanks to its countless health promoting and disease preventing activities. However, three major drawbacks greatly retard its progress from the lab to clinic usage as a drug: low water solubility, low stability, and low systematic bioavailability caused by quickly gastrointestinal and hepatic metabolism [6]. Nano-drug delivery system is one of the most investigated approaches to address the problems of curcumin. Micelle, liposome, emulsion, lipid particle and some special proteins were reported in the literature to increase the water solubility and bioavailability of curcumin. Even though the nano method achieved great break through, they have some common problems, such as low loading ability, low colloidal stability, highly complicated manufacture procedures, large amount of organic solvent usage, and little or no protection to curcumin under pH and UV challenge, which in summary makes them difficult to be applied in pharmaceutical industry. The second mostly used method is chemically modification of curcumin, usually with the help of hydrophilic

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polymer, such as hyaluronic acid and alginate acid [12, 31]. The chemical modification method may solve part of the problems, but it is possible to compromise the functionalities of curcumin. The third widely used method is solid dispersion in which curcumin is mixed with polymer or other materials, mainly in the amorphous or molecular level mixture to increase the water solubility, dissolution rate and bioavailability [16, 17, 32].

In this study, we fabricated curcumin solid dispersion in the matrix of Eudragit® E PO (Cur@EPO) with the following main objectives: (1) to increase the water solubility of curcumin; (2) to increase the stability of curcumin under the pH challenge and UV irradiation test; (3) to increase the bioavailability through transdermal route test.

3.1 Cur@EPO preparation studies

In our pilot study, we found that dropping curcumin organic solution into stirred distilled water lead to formation of curcumin nanoparticles without the help of surfactant and stabilizer. The size of curcumin nanoparticles were in the range of 66.16±0.16 nm to 79.18±0.77 nm depending on the organic solvent used (Figure S1). This observation was in line with previous studies [26, 33]. Curcumin nanoparticles possess negative zeta potential around -20 mV and relative low PDI which indicated they have unified size distribution. However, the curcumin nanoparticle was unstable whose size increased greatly within 24 h. In fact, after 24 h of storage in room temperature, curcumin nanoparticles would form obvious aggregation. The highly unstable properties of curcumin nanoparticles necessitate a delivery system which can effectively reduce

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recrystallization. Eudragit® E PO was selected based on the understanding that the positively-charged polymer would interact with the negatively-charged curcumin nanoparticles, which would decrease the surface Gibbs free (δG) energy and reduce the surface tension thus stabilize the nascent nanoparticles. The preparation procedure of Cur@EPO was illustrated in the Scheme 1. This strategy is effective as the Cur@EPO solution showed isotropic, transparent appearance and long-term stability when compared with curcumin nanoparticle solution in distilled water (Figure 1). Cur@EPO solution resembled curcumin acetone solution as revealed by the UV-vis absorption survey (Figure S2). They both showed characteristic absorption at about 420 nm but the absorption intensity was much stronger than that of curcumin nanoparticle solution which suggested that curcumin in the matrix of Cur@EPO may behave more like free curcumin molecules in acetone solution rather than aggregated molecules in nanoparticles. This hypothesis was supported by the results of size distribution (Figure S3). Curcumin nanoparticles exhibited unimodal size distribution under 100 nm while Eudragit® E PO and Cur@EPO curve displayed very broad distribution from several nanometers to more than 1000 nanometers. Since, we speculated that in Cur@EPO curcumin was dispersed in the matrix of Eudragit® E PO polymer which contradicted our initial conjecture that curcumin nanoparticles were coated by the polymer.

In the preliminary experiments, we loaded different amount of curcumin into Eudragit® E PO with the weight ratio varies from 4%, 8%, 12%, 16%, 20% and 24% w/w. The loading ability

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and efficiency was presented in Figure S4. Note that true loading ability was lower than that of theoretical loading which might be caused by handling loss during sample preparation and lyophilization [34]. When curcumin was loaded at the ratio of 24%, obvious precipitate was detected. So, considering the loading ability and efficiency, we chose three groups for the following study: Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%, named based on the theoretical loading ability. The measured loading ability for Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20% was 10.70±0.06%, 15.01±0.07% and 16.82±0.06% w/w respectively. For the water solubility, we did not measure the exact value since it was very touchy to distinguish supersaturation from normal saturation of this polymer solution. However, we dissolved more than 20 mg of Cur@EPO 16% in 1 mL sodium acetate buffer solution, pH 4.5, and it was quite stable under the period of observation (3 months). No turbidity and precipitate was observed. It means that about 3 mg of curcumin in equivalent was successfully dissolved in 1 mL aqueous medium. Different solubility values of raw curcumin in water were reported according to various preparation and measure methods. In a recently publication, native curcumin presented solubility of 0.39±0.05 μg/mL [34]. Compared with the reported solubility value, Cur@EPO 16% formulation increased the solubility by at least 7500 folds. The solubility calculated in our study was even comparable to that of in normal organic solvents, thus indicating the superiority of this method [6].

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The morphology characteristics of freshly prepared curcumin nanoparticles and Cur@EPO was observed via TEM. In the TEM images (Figure 2a-1, 2), curcumin nanoparticles exhibited classical spherical shape with relatively unified size distribution. The size was below 10 nm which was much smaller than that of DLS measurement (Table 1). This was a common phenomenon and was attributed to hydration of the surface of nanoparticles by water molecules [26]. In fact, the size determined by TEM was also smaller than previous result [27]. Moreover, the curcumin nanoparticles showed smooth round shape which was also a little different from the previous description as ‘rectangularly shaped’ [26]. The different preparation method and treatment may be responsible for the observed discrepancy. In the TEM pictures of Cur@EPO (Figure 2b, c, d), there were no typical curcumin nanoparticles but large irregular particles with the size varied from several hundred nanometers to thousands of nanometers. The size of Cur@EPO corroborate the results of DLS (in Table 1) and was speculated as curcumin loaded Eudragit® E PO polymer. TEM result correlated well with the hypothesis that in the Cur@EPO, the polymer did not coated around the curcumin nanoparticles, but curcumin was dispersed in the matrix of Eudragit® E PO. Thus, TEM observation results confirmed the feasibility of the fabricated Cur@EPO formulation. It is also notable that in the TEM photographs of Cur@EPO 20% (Figure 2d-1, 2), small crystals with the size around 40 nm were observed. This was recognized as curcumin crystal which should be caused by exceeding the threshold of loading [35]. It is interesting to point out that curcumin crystals in Figure 2d-2 was typical needle like shape which is in agreement with previously report, but is much different from that of curcumin

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nanoparticles in Figure 2a-2 [34]. The evident morphology difference may be owing to the different formation mechanism or molecular arrangement. SEM was further employed to examine the morphology difference between Eudragit® E PO and Cur@EPO powder. In Figure 3a, the Eudragit® E PO polymer had large and irregular shape. While in the images of Cur@EPO powder (Figure 3b, c, d), the surface features were much different. Smaller structures with the size of several hundred nanometers were observed. This may be caused by the freezing drying treatment since similar results were also reported in the literature [36]. There was no significant difference among Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%. The SEM results indicate that the surface area was markedly increased compared with Eudragit® E PO polymer and this would contribute to the quick dissolution of Cur@EPO in aqueous media.

3.2 DSC and XRD studies

DSC was used to probe the melting and crystallinity profile of pristine curcumin powder, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, and Cur@EPO samples. The DSC curve of pristine curcumin showed a single endothermic peak at 178.8℃ indicating the crystal melting point (Figure 4) [37]. Eudragit® E PO showed no special endothermic transition while the physical mixture exhibited an endothermic peak at 171.5℃ which was lower than that of pure curcumin. The endothermic peak of the physical mixture was also broader than that of pristine curcumin which was attributed to the solvent effect of molten polymer [17]. In contrast, all three Cur@EPO samples presented no endothermic peak around the melting point of crystal

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curcumin. This is in agreement with previously reports as in the matrix of solid dispersion, the curcumin crystal was transformed to amorphous state [32, 38].

The X-ray diffraction pattern of the samples were depicted in Figure 5. The result seems contradicted our prediction as all the Cur@EPO samples showed very strong peak at 8.93° 2θ and weaker signals at 17.63°, 18.89°, 24.41°, 26.65°, 26.99°, 30.97°and 35.85° 2θ. While pure curcumin crystal and curcumin Eudragit® E PO physical mixture exhibited peaks at 8.89°, 11.87°, 14.25°, 17.33°, 17.87°, 24.51°, and 27.07° 2θ. The result means that all the Cur@EPO samples contained crystal material. But it seems that the crystal compound was not curcumin since most of the peaks did not match with each other. The crystal compound in Cur@EPO sample was determined as sodium acetate which was confirmed by its XRD pattern showed in Figure S6. The characteristic peaks of sodium acetate matched well with the peaks of Cur@EPO thus strongly suggest that the crystal compound is sodium acetate not curcumin. Sodium acetate should be introduced from the sodium acetate buffer solution during the lyophilization treatment. Similar phenomenon has also been reported in the previously researches [39, 40]. 3.3 FT-IR and 1H NMR analysis

The possible interaction between curcumin and Eudragit® E PO was studied through FT-IR and 1

H NMR analysis. Even though curcumin possesses extremely low solubility in aqueous media,

with the help of some polymers, curcumin could be well dissolved in water [41]. However, the mechanism of the increased solubility, and interaction between curcumin and the polymers were

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not well elucidated. In our pilot study, we chose Eudragit® E PO based on the understanding that the positive charge of this polymer may interact with curcumin since it bears negative charge. And this electrostatic interaction may contribute to the enhanced solubility and stability of curcumin. Nevertheless, the expectation of interaction between amine groups of Eudragit® E PO with curcumin was not supported in the FT-IR result. In Figure 6, FT-IR spectra of curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture and three Cur@EPO samples were depicted. Physical mixture of curcumin with Eudragit® E PO exhibited both the characteristic peaks of curcumin and Eudragit® E PO. The spectra of Cur@EPO samples showed some significant changes. Peak at 3492 cm-1, as assigned to stretch of OH in curcumin, disappeared in the spectra of all the Cur@EPO samples which suggested the involvement of the phenol OH group in the interaction [42]. On the other hand, signal at 1720 cm-1 which was recognized as stretch of carbonyl in the polymer shifted to lower wavelength at 1714 cm-1 in the spectra of Cur@EPO. The intensity also markedly reduced in spectra of Cur@EPO than that of Eudragit® E PO. H bond interaction was accountable for the observed changes [43]. It was also notable that Cur@EPO FT-IR spectra revealed two strong peaks at 1560 cm-1 and 1400 cm-1 which were assigned as the symmetric and asymmetric stretch of carboxylic group [44, 45]. The carboxylic group was determined as belonging to the sodium acetate which was introduced from the buffer solution during the freeze drying treatment. In solid dispersion formulation, H bonding was usually determined as the possible interaction between curcumin and excipients [43, 46].

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FT-IR results also suggest that the formation of H bonding involved the carbonyl group in the Eudragit® E PO polymer that accepted the H atom from OH group of curcumin. 1

H NMR spectroscopy result confirmed the finding of FT-IR spectra (Figure S5). The signals of

chemical shift were assigned according to the previously publication [47]. From the result, no significant changes were presented since the interaction between curcumin and polymer was weak H bonding interaction. In the spectra of Cur@EPO, weak signals assigned to curcumin were revealed. The signals of curcumin in Cur@EPO was widened compared with the signals of pure curcumin. This has been reported in the publication as proof of the interaction between curcumin and the polymer matrix [40]. Furthermore, chemical shifts in Cur@EPO were slightly different from those of Eudragit® E PO because the interaction could influence the electron density of the polymer as well as the curcumin [48]. The signals of polymer in Cur@EPO samples (especially a, b, c, d and e illustrated in Figure S5) shifted with small range to lower values (shielding) resulted from the H bonding formation with the OH group of curcumin. Furthermore, signals of curcumin in Cur@EPO samples shifted to higher values (deshielding) which was also resulted from the H bonding interaction with polymer.

Taking above evidence into account, we hypothesized the possible interaction between curcumin and Eudragit® E PO in the solid dispersion which was illustrated in Scheme 2. Loading ability test indicated that 20% w/w curcumin loading would not be problematic but the TEM result clearly showed that at this loading level the polymer could not hold all the curcumin, thus lead

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the formation of small curcumin crystals. So the threshold level for curcumin loading in Cur@EPO should be within the range of 16% to 20% w/w. Considering the molecular weight of Eudragit® E PO repeating unit (385) and curcumin (368), about 6 polymer repeating units were used to stabilize one curcumin molecule. Since FT-IR spectra implied that all phenol OH groups involve in the interaction, plus the fact that positive charged amine group in the polymer may contribute to the interaction which was an interesting common point but without explicit demonstration in several previous works [17, 49-51], the carbonyl group in the dimethylaminoethyl segment was believed as the H bond accepter as depicted in Scheme 2.

3.4 Stability tests

Curcumin is unstable and quickly degraded in both neutral and alkaline solution. Many researches tried to solve the water solubility problem of curcumin but few of them attempted to increase its chemical stability. To this regard, the protection ability of Cur@EPO formulation in different pH values was investigated. The result indicated that under all pH values examined (5.0, 6.0, 7.0 and 8.0), Cur@EPO can effectively protect curcumin from either hydrolysis or separation out in the form of precipitate (Figure 7). It is notable that even in acidic PBS, the absorptions of pure curcumin solution also tend to decrease. This phenomenon is in line with the previous results and can be explained by the low water solubility of curcumin in aqueous solution which lead to the formation of crystal and consequently decreased the absorption [32]. In PBS pH 8.0, the absorption value of pure curcumin solution quickly decreased, whereas all the

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Cur@EPO sample solutions maintained more than 90% of the initial absorption values. In the pH challenge test, the polymer not only increased the water solubility of curcumin but also effectively delayed the crystallization and decomposition. In the solid dispersion formulations, polymer can protect the loaded drug from hydrolysis and similar results were also well recognized in the literature [52].

UV irradiation is an effective and simple sterilization method both in the food and pharmaceutical industry [53]. Unfortunately, curcumin decomposes quickly under the UV treatment and this weakness requires the formulation to offer UV irradiation protection to curcumin. Direct and indirect UV photolysis were conducted according to our previous study [54]. In both the direct and indirect UV photolysis results (Figure 8), it is evident that Cur@EPO protected the curcumin from degradation. In direct photolysis (Figure 8a), 24 h of UV treatment destroyed about half of the curcumin in its raw form. In contrast, in Cur@EPO samples, more than 85% of curcumin was preserved. Different Cur@EPO sample demonstrated certain variations but the difference was not considered as significant. In the indirect UV irradiation (in the solution form), similar pattern was shown (Figure 8b). But here, we cannot exclude the possible effect of the solvent since curcumin is only dissolved in organic solvent. Still, along with the direct UV photolysis result, we concluded that the polymer in Cur@EPO could effectively protect the curcumin against UV degradation. In fact, after 24 h of UV treatment in

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indirect photolysis, the curcumin UV absorption peak in ethanol was totally disappeared which was in markedly contrast with the Cur@EPO solution (Figure S7).

3.5 Drug release profile

Considering the fact that small curcumin crystal was detected in Cur@EPO 20% which may render it unstable in the application, we used Cur@EPO 16% in the release and transdermal test. Curcumin release profile under different pH values were investigated in PBS containing 1% w/w Tween 80 (Figure 9). Release pattern implied that pH value influenced the release profile that lower pH accelerated release and higher pH reduced the release speed. The nature of interaction between curcumin and Eudragit® E PO is accountable for the release characteristic as H bond is highly influenced by the pH of the aqueous media [55]. The release profile was monitored in 48 h and it was notable that curcumin in Cur@EPO 16% released in a sustained pattern as partial (from 7.6% to 32.5%) of curcumin was released depending on the different pH values. The pH dependent and sustained release of the fabricated curcumin solid dispersion would be an important merit in the practical applications [56].

3.6 In vitro transdermal test

Transdermal administration route may provide an alternative to curcumin but only few researches focused on this issue. Curcumin nanoemulsion was prepared for in vitro transdermal test using shed snake skin [19]. Anti-inflammatory effect of several matrix-type transdermal

24

films prepared by curcumin and selected polymers were investigated in vitro and in vivo tests [20]. On the other hand, Eudragit® E 100 was reported to be formulated as transdermal drug-in-adhesive patches [57]. We assessed the feasibility of Cur@EPO 16% as transdermal delivery vehicle and the result was presented in Figure 10. Curcumin DMSO solution and freshly prepared curcumin nanoparticles solution were used as control groups. It is well known that DMSO greatly enhance the skin permeation of hydrophobic drugs [58]. In our study, curcumin DMSO solution also demonstrated significant higher permeation speed than that of Cur@EPO 16% solution. On the other hand, even though curcumin nanoparticles maintained the size below 200 nm at least within 10 h (Figure S1), no curcumin was detected in the receiver compartment. Cur@EPO 16% solution achieved plateau permeation at about 9 h with around 6.5 μg/cm2. The permeation level of Cur@EPO 16% was lower than that of curcumin DMSO solution but comparable with that of permeation profile obtained via curcumin nanoemulsion and suggest the possible application of this formulation in transdermal delivery of curcumin [19].

4. Conclusion

Solid dispersion fabricated by curcumin and Eudragit® E PO using a simple solution mixing method was successfully formulated. Water solubility of curcumin in Cur@EPO was significantly increased to at least 3 mg/mL which was similar to that in conventional organic solvents. UV absorption survey, TEM, DSC and XRD measurement indicate that curcumin was dispersed in the matrix of polymer in amorphous state. The interaction between curcumin and the

25

polymer was investigated by FT-IR and 1H NMR the results of which suggest OH group in curcumin and carbonyl group in the polymer involved in the H bonding formation. Moreover, the stability of curcumin was markedly enhanced in Cur@EPO as examined by pH challenge and UV irradiation test. The H bonding between curcumin and the polymer warranted the high water solubility as well as sustained release which is an important merit in practical application. At last, in vitro transdermal test was carried out to verify the usefulness of the formulation to delivery curcumin through this promising administration route. Collectively, Cur@EPO was prepared by a simple method and proved to be an effective formulation to circumvent the drawbacks of curcumin, and at the same time keep its therapeutic efficacy.

5. Acknowledgements

This research was supported by Kyung-Nong Company, the International Research & Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. 2012K1A3A1A20031356), a Korea University grant and Institute of Biomedical Science & Food Safety, Korea University Food Safety Hall.

Supplementary data:

26

Size, PDI and zeta potential changes within 10 h of curcumin nanoparticles prepared by acetone, ethanol and methanol curcumin solution mixing with distilled water (Figure S1), UV-vis absorption survey of curcumin solution in acetone, curcumin-Eudragit® E PO solid dispersion solution in sodium acetate buffer solution, pH 4.5, and curcumin nanoparticle solution immediately after preparation (Figure S2), size distribution of curcumin nanoparticles in distilled water, Eudragit® E PO and Cur@EPO in acetic acid buffer pH 4.5 (Figure S3), loading ability and efficiency of curcumin-Eudragit® E PO solid dispersion (Figure S4), X-ray diffraction (XRD) pattern of sodium acetate (Figure S5), 1H NMR spectra of pristine curcumin, Eudragit® E PO, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20% (Figure S6) and UV-vis absorption survey of curcumin ethanol solution and Cur@EPO acetic acid buffer before and after indirect UV photolysis treatment (Figure S7).

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Captions to Figures:

Scheme 1. Preparation procedure for curcumin-Eudragit® E PO solid dispersion.

Figure 1. Appearance comparison of a: curcumin solution in acetone; b: curcumin-Eudragit® E PO solid dispersion solution in sodium acetate buffer solution, pH 4.5; and c: curcumin nanoparticle solution immediately after preparation.

Figure 2. Transmission electron microscopy (TEM) images of curcumin nanoparticles (a-1, scale bar is 100 nm; a-2, scale bar is 20 nm), Cur@EPO 12% (b-1, scale bar is 100 nm; b-2, scale bar is 20 nm), Cur@EPO 16% (c-1, scale bar is 100 nm; c-2, scale bar is 20 nm), and Cur@EPO 20% (d-1, scale bar is 50 nm; d-2, scale bar is 20 nm). Note in d-1 and d-2, red arrows point to curcumin crystals.

Figure 3. Scanning electron microscope (SEM) images of Eudragit® E PO (a), Cur@EPO 12% (b), Cur@EPO 16% (c) and Cur@EPO 20% (d).

Figure 4. Differential scanning calorimetry (DSC) results of pristine curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, Cur@EPO 12% , Cur@EPO 16% and Cur@EPO 20%.

Figure 5. X-ray diffraction (XRD) results of pristine curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%.

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Figure 6. Fourier transform infrared spectroscopy (FT-IR) spectra of pristine curcumin, Eudragit® E PO, curcumin and Eudragit® E PO physical mixture, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%.

Scheme 2. Interaction between Eudragit® E PO polymer and curcumin molecule, bold dash line means H bond.

Figure 7. Stability test of curcumin, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20% in PBS pH 5.0, 6.0, 7.0 and 8.0.

Figure 8. a, direct UV photolysis of curcumin, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20% in 24 h; b, indirect UV photolysis of curcumin, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20% in 24 h.

Figure 9. Release profile of Cur@EPO 16% in HCl 0.1 N, PBS pH 4.5, 6.5, 7.4 release media containing Tween 80 1 wt %.

Figure 10. Cumulative curcumin permeation on synthetic membranes (Strat-M) of curcumin DMSO solution, Cur@EPO sodium acetate buffer solution and fresh prepared curcumin nanoparticle solution.

Figure S1. Size, PDI and zeta potential changes within 10 h of curcumin nanoparticles prepared by acetone, ethanol and methanol curcumin solution mixing with distilled water.

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Figure S2. UV-vis absorption survey of curcumin solution in acetone, curcumin-Eudragit® E PO solid dispersion solution in sodium acetate buffer solution, pH 4.5, and curcumin nanoparticle solution immediately after preparation.

Figure S3. Size distribution of curcumin nanoparticles in distilled water, Eudragit® E PO and Cur@EPO in acetic acid buffer pH 4.5.

Figure S4. Loading ability and efficiency of curcumin-Eudragit® E PO solid dispersion.

Figure S5. X-ray diffraction (XRD) pattern of sodium acetate. Figure S6. 1H NMR spectra of pristine curcumin, Eudragit® E PO, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%.

Figure S7. UV-vis absorption survey of curcumin ethanol solution and Cur@EPO acetic acid buffer before and after indirect UV photolysis treatment.

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35

36

37

38

39

40

41

42

43

44

45

46

Table 1 Physicochemical parameters of curcumin nanoparticle, Cur@EPO 12%, Cur@EPO 16% and Cur@EPO 20%

PDI

Zeta potential (mV)

Loading ability (%)

Loading efficacy (%)

Stabilityc

-

-

Not stable

Sample

Size (nm)

Curcumin nanoparticlea

66.16±0.16

0.21±0.05 -17.23±1.66

Cur@EPO 12%b

450.23±9.02

0.40±0.07

44.40±1.08

10.70±0.06 92.79±1.45

Stable

Cur@EPO 16%

433.10±12.90 0.60±0.08

43.33±1.76

15.01±0.07 97.60±1.70

Stable

Cur@EPO 20%

475.70±12.15 0.57±0.05

43.6±1.25

16.82±0.06 87.50±1.56

Stable

a

Curcumin nanoparticle solution was prepared by dropping curcumin acetone solution into distilled water under stirring at 1500 rpm b

Cur@EPO was prepared according to the method described in section 2.2

c

Stability was examined by turbidity, transparency and sediment check

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Graphical abstract

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