Development and evaluation of a novel phytosome-loaded chitosan microsphere system for curcumin delivery

International Journal of Pharmaceutics 448 (2013) 168–174

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Development and evaluation of a novel phytosome-loaded chitosan microsphere system for curcumin delivery Jifen Zhang ∗ , Qin Tang, Xiaoyu Xu, Na Li College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, China

a r t i c l e

i n f o

Article history: Received 25 November 2012 Received in revised form 3 February 2013 Accepted 13 March 2013 Available online 19 March 2013 Keywords: Curcumin Phytosomes Chitosan microspheres Sustained-release Lipophilic compound

a b s t r a c t In this study, we developed a novel drug delivery system, curcumin-phytosome-loaded chitosan microspheres (Cur-PS-CMs) by combining polymer- and lipid-based delivery systems. Curcumin exhibits poor water-solubility and is rapidly eliminated from the body. We aimed to use our novel delivery system to improve the bioavailability and prolong the retention time of curcumin in the body. The Cur-PS-CMs were produced by encapsulating curcumin-phytosomes (Cur-PSs) in chitosan microspheres using ionotropic gelation. The final microsphere was spherical, with a mean particle size of 23.21 ± 6.72 ␮m and drug loading efficiency of 2.67 ± 0.23%. Differential scanning calorimetry and Fourier transform infrared spectroscopy demonstrated that the integrity of the phytosomes was preserved within the polymeric matrix of the microspheres. The in vitro release rate of curcumin from the Cur-PS-CMs was slower than that from curcumin-loaded chitosan microspheres (Cur-CMs) in pH 1.0, 4.0, 6.8, and 7.4. Pharmacokinetic studies in rats dosed with Cur-PS-CMs showed a 1.67- and a 1.07-fold increase in absorption of curcumin compared with Cur-PSs and Cur-CMs, respectively. The half-life of curcumin orally administration of Cur-PS-CMs (3.16 h) was longer than those of Cur-PSs (1.73 h) and Cur-CMs (2.34 h). These results indicated that the new Cur-PS-CMs system combined the advantages of chitosan microspheres and phytosomes, which had better effects of promoting oral absorption and prolonging retention time of curcumin than single Cur-PSs or Cur-CMs. Therefore, the PS-CMs may be used as a sustained delivery system for lipophilic compounds with poor water-solubility and low oral bioavailability. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Curcumin, a hydrophobic polyphenol derived from the rhizome of Curcuma longa L. (Zingiberaceae or dietary turmeric) has been shown to exhibit antioxidant, anti-inflammatory, antimicrobial, anti-amyloid, and antitumor activities (Maheshwari et al., 2006; Anand et al., 2008; Srivastava et al., 2011). In addition, the nontoxic nature of curcumin has been demonstrated by its long history of dietary use and clinical trials (Cheng et al., 2001; Sharma et al., 2004). To date, there are no studies that show toxicity associated with the use of curcumin, even at very high doses. Despite its promising therapeutic efficacy and favorable safety profile, the clinical application of curcumin has been obstructed by its poor solubility in water, rapid half-life, and low bioavailability after oral administration. Its maximum solubility is 11 ng/mL in aqueous buffer (pH 5.0) (Tonnesen et al., 2002). Following oral administration (up to 8 g/day), only a trace amount of curcumin was detected in blood. In addition, the oral bioavailability was only 1% in rats (Pan et al., 1999; Yang et al., 2007). Curcumin undergoes a

∗ Corresponding author. Tel.: +86 23 68251225; fax: +86 23 68251048. E-mail addresses: [email protected], [email protected] (J. Zhang). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.03.021

very high first-pass metabolism and is eliminated rapidly. Thus, its retention time in circulation is very short (Pan et al., 1999; Sharma et al., 2007; Yang et al., 2007). To overcome these limitations, various formulations and techniques have been investigated over the past decades. Such studies included the use of solid dispersions (Paradkar et al., 2004), complex formation with cyclodextrins (Yallapu et al., 2010), copolymeric micelles (Song et al., 2011), polymeric nanoparticles (Shaikh et al., 2009; Das et al., 2010; Anitha et al., 2011; Kim et al., 2011), lipid-based nanoparticles (Sou et al., 2008; Dadhaniya et al., 2011), liposomes (Chen et al., 2009), a phospholipid complex (Maiti et al., 2007), and self-microemulsion (Cui et al., 2009; Setthacheewakul et al., 2010). However, to our knowledge, these techniques were used alone and only addressed one of the shortcomings of curcumin. For example, the curcumin-phospholipid complex increased the AUC of curcumin by 3.37 times. However, it could only lengthen the half-life of curcumin from 1.45 h to 1.96 h. Several studies have shown that an appropriate combination of different carrier systems can achieve the advantages of each system while avoid the shortcomings. Feng et al. (2004) fabricated a novel drug delivery device called liposomes-in-microsphere (LIM), in which liposomes could protect loaded therapeutic proteins from the harsh conditions involved in the subsequent

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fabrication process, as well as the acidic microenvironment inside the PLA-PEG-PLA microspheres. Cai et al. (2012) formulated the huperzine A–phospholipid complex and loaded the complex into PLGA–PEG–PLGA gel in order to reduce the burst effect of the gel and control the huperzine A release. To encapsulate hydrophilic insulin into strong lipophilic poly (hydroxybutyrateco-hydroxyhexanoate) (PHBHHx), insulin was first complexed with phospholipids to enhance the lipophilicity and then loaded into PHBHHx nanoparticles (Peng et al., 2012). To extend curcumin delivery time using a phospholipid complex, phytosomes (PSs), or self-assembling bilayer vehicles of phospholipid complex, were further encapsulated within polymeric microspheres. The main purpose of the microspheres was to control the exposure of phytosomes, and consequently provide sustained release of a drug. Chitosan was chosen because of its unique biological properties, including favorable biocompatibility, biodegradability, polycationicity, and mucoadhesiveness (Dasha et al., 2011). Chitosan can enhance the absorption of drugs into gastric mucosa by mucoadhesion or by opening tight junctions between epithelial cells (Senel et al., 2000; Thanou et al., 2001). The positive charge of chitosan makes it suitable to combine with negatively charged PSs. The preparation of chitosan microspheres is entirely aqueous and should not affect phytosome stability. The aim of our study was to prepare a combined drug delivery system to promote the absorption and slow down the elimination of curcumin after oral administration. The phytosomes and chitosan microspheres were integrated to fabricate a new vehicle in which both components could promote the absorption of curcumin. In addition, the matrix of the microspheres could delay the release of curcumin. The new curcumin-phytosome-loaded chitosan microspheres (Cur-PS-CMs) were prepared by encapsulating curcumin-phytosomes (Cur-PSs) in chitosan microspheres. We then characterized the Cur-PSs and Cur-PS-CMs. We determined the in vitro drug release behavior of the microspheres at different pH levels to demonstrate the mechanism of action of the combined drug delivery system. In addition, the pharmacokinetics was evaluated in rats after oral administration of natural Cur, Cur-PSs, curcumin-loaded chitosan microspheres (Cur-CMs), and Cur-PS-CMs.

2. Materials and methods

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2.3. Preparation of Cur-PS-CMs The Cur-PS suspension was diluted with 2% (w/v) acetic acid until the final curcumin concentration was 0.1 mg/mL. Chitosan (400 mg) was dissolved in 200 mL of the diluted suspension. The resulting solution was then fed through a 2 mm diameter nozzle via peristaltic pump (BT-100, Shanghai Huxi Analysis Instrument Factory Co., Ltd., China) at a flow rate of 2.0 mL/min and dropped into 400 mL of strongly agitated sodium tripolyphosphate (TPP) solution (0.3%, w/v). The resulting chitosan microspheres were left in the dark overnight. The TPP solution was then decanted. The microspheres were washed several times with deionized water, laid out on aluminum trays, and oven dried at 40 ◦ C until their weight remained constant. Cur-CMs and blank chitosan microspheres (CMs) were prepared using the procedures just described, except a curcumin suspension or 2% (w/v) acetic acid with no solute was used instead of the Cur-PS suspension. 2.4. Characterization of Cur-PSs and Cur-PS-CMs 2.4.1. Complex formation efficiency and drug loading The percentage of curcumin complexed with phospholipids was determined as follows: Cur-PS suspension was diluted 1-fold with 0.5% (w/v) Tween-80 and then centrifuged at 30,000 rpm for 2 h at 4 ◦ C. The supernatant was isolated and the amount of free curcumin was determined by UV/Vis spectroscopy at 420 nm. To determine the total amount of curcumin, 0.1 mL of the Cur-PS suspension was diluted in proper methanol, adjusting the volume to 50 mL. The complex formation efficiency was calculated according to the following formula: Complex formation efficiency (%) =

 Total amount of curcumin − amount of free curcumin  Total amount of curcumin

× 100 To determine the loading capacity of the microspheres, 10 mg of microspheres were added to 5 mL of 0.1 mol/L HCl and sonicated for 20 min in a water bath. One milliliter of the suspension was immediately withdrawn and diluted to 10 mL with methanol. After filtration through 0.45 ␮m Millipore filters, the samples were analyzed by UV/Vis spectroscopy at 420 nm.

2.1. Materials Soybean phospholipids (94% purity) were purchased from Shanghai Taiwei Co., Ltd (Shanghai, China). Chitosan (viscosity of 300 cps and deacetylation degree of 93%) was purchased from Golden-shell Biochemical Co., Ltd. (Qindao, China). Curcumin (98% purity) was purchased from Xi’an Rongsheng Biotechnology Co., Ltd. (Xi’an, China). Emodin (98% purity) was purchased from Chroma-standard Medical technology Co., Ltd. (Tianjing, China). Other solvents and chemicals were of analytical or chromatographic grade.

2.2. Preparation of Cur-PSs The Cur-PSs were prepared as previously reported (Maiti et al., 2007), with slight modifications. Briefly, 20 mL of absolute alcohol was mixed with 100 mg of curcumin and 214 mg of soybean phospholipids in a 100 mL round bottom flask. The mixture was stirred at 50 ◦ C for 2 h. Afterwards, the solution was added to 40 mL of 2% (w/v) acetic acid. The resulting mixture was continuously stirred at 50 ◦ C until the odor of alcohol was no longer apparent. All procedures were protected from light.

2.4.2. Particle size and morphology The average diameter and zeta potential of the Cur-PSs were both measured using a Zetasizer ZEN 3600 (Malvern Instruments Ltd., UK) at a fixed scattering angle of 90◦ at 25 ◦ C. The average diameter of the microspheres was measured by laser diffraction using a Malvern Mastersizer 2000 particle sizer (Malvern Instruments Ltd., UK) at a fixed scattering angle of 90◦ at 25 ◦ C. The morphology of the Cur-PSs was analyzed by atomic force microscopy (AFM; SPM, CE Ltd., USA) and transmission electron microscopy (TEM; H-7500, Hitachi Scientific Instruments Ltd., Japan). The suspension was diluted 10-fold with deionized water. For AFM, one drop of suspension was placed on freshly cleaved mica. The sample was air-dried at room temperature and mounted on the microscope scanner. The shape was observed and imaged in tapping mode. For TEM, one drop of suspension was placed on a 400 mesh copper grid coated with carbon. After 20 min, the grid was tapped with filter paper to remove surface water and stained using a solution of phosphotungstic acid (2%, w/v) for 20 min. Then the stained sample was dried in air and the morphology was observed. The morphology of Cur-PS-CMs was evaluated by scanning electron microscopy (SEM). The microspheres were suspended in absolute alcohol and sonicated for 5 s to break up the aggregates

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without destroying the microspheres. One drop of suspension was spread onto an aluminum stub covered with double-sided adhesive tabs. After the alcohol evaporated completely, the microspheres were vacuum-coated with a gold-palladium film and directly analyzed with SEM (S-3400N, Hitachi Scientific Instruments Ltd., Japan). 2.4.3. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy For these studies, Cur-PSs were lyophilized (CoolSafe 110-4 Pro, ScanLaf Ltd., Denmark) to obtain a dry sample. Two groups of samples were compared. One group consisted of the natural curcumin, soybean phospholipids, Cur-PSs, and a physical mixture of curcumin and phospholipids. The other group consisted of the natural curcumin, blank CMs, a physical mixture of curcumin and blank CMs, Cur-CMs, and Cur-PS-CMs. DSC analysis was performed using a DSC 200PC (Netzsch Ltd., Germany). With approximately 10 mg of sample sealed in an aluminum pan, the thermal behavior was analyzed from 50 ◦ C to 250 ◦ C using a heating rate of 10 ◦ C/min. To investigate the interaction of curcumin and phospholipids in Cur-PSs, FTIR spectra were measured using a FTIR microscope (Spectrum GX, PE Ltd., USA). Comparable data was obtained by averaging 50 scans of data between 4000 cm−1 and 400 cm−1 (scanning speed of 4 cm−1 ). Natural curcumin, soybean phospholipids, the mixture of curcumin and phospholipids, and the Cur-PSs were analyzed. 2.5. In vitro drug release studies In vitro curcumin release profiles from Cur-CMs and Cur-PS-CMs were determined at pH 1.0, 4.0, 6.8, and 7.4. Multiple releasing mediums were used, including 0.1 mol/L HCl (pH 1.0), 0.01 M citric acid-disodium hydrogen phosphate buffer (pH 4.0), and 0.01 M phosphate-buffered saline solution (pH 6.8 and pH 7.4) containing 0.5% (w/v) Tween-80. A suitable amount of microspheres containing about 0.5 mg of curcumin were placed in an Eppendorf tube and 5 mL of releasing medium was added. Nine Eppendorf tubes were used in order to account for each time interval, specifically 0, 2, 5, 8, 12, 18, 24, 36, and 48 h. All samples were incubated at 37 ◦ C under gentle agitation in the dark. At the desired times, the samples were centrifuged at 15000 rpm for 10 min and the supernatants were discarded. The remaining amount of curcumin in the microspheres were first extracted using 0.1 mol/L HCl and methanol, and quantified using a spectrophotometer at 420 nm. The experiments were carried out in triplicate. 2.6. Pharmacokinetic studies in vivo 2.6.1. Oral administration For our in vivo pharmacokinetic studies, Sprague Dawley (SD) rats weighing 300–350 g were divided into four groups (n = 5). Group 1, Group 2, Group 3, and Group 4 were administered natural curcumin, Cur-PSs, Cur-CMs, and Cur-PS-CMs, respectively, by oral gavage. The curcumin dose for all cases was fixed at 100 mg/kg body weight. The blood samples (0.5 mL) were collected and placed into heparin-rinsed tubes at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10, and 12 h after oral administration. Samples were preserved at −80 ◦ C. 2.6.2. Sample preparation Plasma was separated by centrifugation of the blood samples at 5000 rpm for 10 min at 4 ◦ C. Internal standard solution (25 ␮L containing 2.45 ␮g/mL of emodin dissolved in methanol) was added to 200 ␮L of plasma. Extraction of curcumin was accomplished by adding 500 ␮L of ethyl acetate to the diluted plasma and vortexing for 3 min. The samples were then centrifuged at 10,000 rpm

for 10 min and the supernatants were set aside. Extraction was repeated by adding another 500 ␮L of ethyl acetate to the residue. Both supernatants were combined and dried under a stream of nitrogen gas at 30 ◦ C. The residues that remained were resuspended in 100 ␮L of methanol and centrifuged at 10,000 rpm for 10 min. Afterwards, 20 ␮L of the supernatants were analyzed using HPLC method. 2.6.3. Quantification of isolated curcumin Curcumin isolated from plasma was assayed via reversed phaseHPLC with UV detector (Agilent 1200, USA) using an Agilent SB-C18 ODS column (5 ␮m, 4.6 mm × 250 mm) at 30 ◦ C. The mobile phase solution was composed of 55:45 acetonitrile:2% (w/v) acetic acid. The flow rate was set at 1.0 mL/min and the detection wavelength used was 420 nm. The retention times for curcumin and emodin were about 6.8 min and 10.8 min, respectively. Linearity was obtained from 5.5 to 440 ng/mL. The coefficient of variation for intra- and inter-day assays was less than 10%. The average recovery of curcumin from the isolated plasma solution was greater than 90%, and 82.3% of curcumin was extracted from blood. 2.6.4. Pharmacokinetic application and statistics Pharmacokinetic calculations were performed on each individual set of data using DAS 3.2 (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The pharmacokinetic results were represented as the mean ± standard error of the mean (S.E.M.). One-way ANOVA was performed to analyze differences among groups with SPSS (Version 10.0). 3. Results and discussion 3.1. Complex formation efficiency and drug loading In this study, a colloidal suspension of Cur-PSs was formed, in which 90.81 ± 1.32% of curcumin was complexed with phospholipids. The Cur-PS-CMs were fabricated by ionotropic gelation, using TPP as the cross-linking agent. The drug loading efficiency of Cur-PS-CMs was 2.67 ± 0.23% (n = 3), which was lower than that of Cur-CMs (5.37 ± 0.48%). 3.2. Particle size and morphology Dynamic light scattering showed that the mean diameter of Cur-PSs was 68.59 ± 10.16 nm (n = 3), which correlated with the particle sizes observed by AFM (63.35 nm) and TEM (75.41 nm). The negative surface charge of the particles (−12.6 ± 4.1 mV; n = 3) was suitable for encapsulating Cur-PSs into CMs because chitosan is positively charged. The TEM and AFM images (Fig. 1) indicated that the Cur-PSs were spherical or ellipsoidal, self-closed structures. This demonstrated that the complex formation of curcumin and phospholipids did not change the amphipathic properties and water dispersion associated with phospholipids. Upon interaction with water, the hydrophilic heads of the phospholipids oriented toward the water compartment and the lipophilic tails oriented away from the water. In this way, PS, a lipid bilayer vesicle, was formed (Saraf, 2010). The concentrations of chitosan and TPP have significant effect on the size of chitosan microspheres. In this study, 0.25% (w/v) chitosan solution and 0.3% (w/v) TPP solution were chosen in order to obtain small and uniform microspheres, which were demonstrated by SEM. The Cur-PS-CMs were spherical and heterogeneous in size (Fig. 2). Their diameters ranged from 13 to 34 ␮m, with the mean diameter equal to 23.21 ± 6.72 ␮m (n = 3) as measured by laser diffraction. The magnified image of a single microsphere

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Fig. 1. Mean particle size of Cur-PSs measured by laser diffraction (a) and images of Cur-PSs viewed under TEM (b), AFM (c) and three-dimensional view of AFM image (d).

showed that the surface was porous and loose with many thin fibers crossing, which might be TPP cross-linked with chitosan. 3.3. DSC and FTIR DSC analysis was performed to confirm the presence of curcumin in the phytosomes. As shown in the DSC thermogram (Fig. 3), there was a broad endothermic peak of phospholipids at 145.8 ◦ C. The sharp endothermic peak of natural curcumin was found approximately at 175.7 ◦ C. The mixture of curcumin and phospholipids showed two peaks: one at 140.9 ◦ C and the other at 164.0 ◦ C. Both peaks were lowered slightly, which may be due to the natural hydrophobic interaction between curcumin and phospholipids suggested by Began et al. (1999). The thermogram of Cur-PSs exhibited a single peak at 125.5 ◦ C, and the characteristic peaks of curcumin and phospholipids were not observed. This suggested that the Cur-PSs were not simple mixtures of curcumin and phospholipids. The physical state of curcumin in the microspheres was also examined by DSC. Blank CMs and curcumin showed individual endothermic peaks at 101.6 ◦ C and 175.7 ◦ C, respectively (Fig. 3), due to their melting points. We observed strong endothermic peaks

of CMs at 101.8 ◦ C and weak endothermic peaks of curcumin at 166.0 ◦ C for the mixture of blank CMs and curcumin. Cur-CMs had a similar thermogram to the mixture, with the endothermic peaks of CMs and curcumin at 107.6 ◦ C and 170.5 ◦ C, respectively. This similarity indicated that, when curcumin was loaded directly into CMs, the physical state of curcumin did not change, and curcumin still existed in microcrystal form. However, in the case of Cur-PS-CMs, the prominent peak belonging to curcumin at 175.7 ◦ C completely disappeared, and only the melting point peak of CMs remained at 102.7 ◦ C. This could be due to the complete and tight complex formation of curcumin with phospholipids, which prevented the dissociation of curcumin from Cur-PS-CMs during the preparation process. We used FTIR to confirm the interaction between curcumin and phospholipids. In the FTIR spectra (Fig. 4), the band at 3413 cm−1 was attributed to the stretching vibration of the phenolic-OH group in natural curcumin. Additionally, sharp absorption bands were observed at 1603 cm−1 (stretching vibrations of a benzene ring), 1510 cm−1 (C O and C C vibrations), and 1278 cm−1 (aromatic C O stretching vibration). The strong peaks at 1737 cm−1 and 1236 cm−1 in phospholipids were due to C O absorption and P O absorption, respectively. The strong peaks at 2925 cm−1 and

Fig. 2. SEM photographs of (a) Cur-PS-CMs; (b) surface of single Cur-PS-CM.

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Fig. 3. DSC thermogram of (a) (i) phospholipids, (ii) curcumin, (iii) Cur-PSs, and (iv) the mixture of phospholipids and curcumin; (b) (i) blank CMs, (ii) curcumin, (iii) the mixture of curcumin and blank CMs, (iv) Cur-CMs, and (v) Cur-PS-CMs.

2854 cm−1 and the weak peak at 1376 cm−1 in phospholipids could be due to stretching and deformation of methyl groups. The peak at 1465 cm−1 observed in phospholipids could be due to bending vibration of CH2 . These peaks were found, not shifted, when analyzing the mixture of curcumin and phospholipids. This suggested that there was no interaction between curcumin and phospholipids. A shift from 3413 cm−1 to 3263 cm−1 compared with natural curcumin, and a shift from 1236 cm−1 to 1240 cm−1 compared with phospholipids were exhibited by Cur-PSs. Additionally, the peak at 3263 cm−1 became wider. These changes indicated that curcumin and phospholipids formed a complex by hydrogen bonding between the OH group of the phenol rings of curcumin and the P O group of the phospholipids. This interaction lowers the phase transition temperature of phospholipids from 145.8 ◦ C to 125.5 ◦ C and makes the second sharp peak of phospholipids disappear. The interaction of curcumin with the polar part of the phospholipids makes the long hydrocarbon tail of the latter bend freely and envelope its own polar head. Thus, the sequence of the phospholipids is interrupted (Maiti et al., 2007). 3.4. Drug release in vitro In order to analyze the mechanism of action of our Cur-PS-CMs, curcumin release from Cur-CMs and Cur-PS-CMs in vitro at different

Fig. 4. FTIR spectra of (i) phospholipids, (ii) curcumin, (iii) Cur-PSs, and (iv) the mixture of phospholipids and curcumin.

pH were compared. Tween-80 aqueous solution (0.5%) was adopted to reach sink conditions. At each time, the remaining curcumin in the microspheres was quantified to calculate the percentage of released compound according to Das et al. (2010) because of the instability of curcumin in acid and alkaline pH. Curcumin was rapidly released from Cur-CMs within 5 h, but was released at a more sustained rate in pH 1.0, 4.0, 6.8, and 7.4 (Fig. 5). The quick release within 5 h was barely affected by the pH of the releasing medium because the released drug was originally absorbed in or close to the loose surface of the microspheres. The release rate after 5 h slightly increased as the pH of the releasing medium decreased. In the subsequent 43 h, about 39%, 35%, 32%, and 31% of the curcumin was released from Cur-CMs in pH 1.0, 4.0, 6.8, and 7.4, respectively. The pH-dependent release can be attributed to the swelling of the chitosan matrix. In acidic medium, chitosan can swell and gradually dissolve due to the protonation of residual amine groups. The release pattern of curcumin from Cur-PS-CMs was similar to that from Cur-CMs except that it was slower. After 5 h, about 39–43% of the curcumin was released from Cur-PS-CMs, which was significantly lower than 59–61% released from Cur-CMs. This was probably due to two reasons: (1) the force of attraction between the negative charge of PSs and the positive charge of chitosan inhibited the diffusion of PSs from the microspheres; and (2) curcumin was complexed with phospholipids, requiring some time for curcumin to release from Cur-PSs. From 5 h to 48 h, about 27%, 28%, 30%, and 29% of the curcumin was released from Cur-PS-CMs in pH 1.0, 4.0, 6.8 and 7.4, respectively. This was lower than the release behavior of Cur-CMs, especially in acidic medium. The Cur-PS-CMs remained intact for 48 h, and no obvious swelling or degradation was observed. This may be because the residual amine groups of chitosan were occupied by PSs, so the protonation of these amine groups was limited. Thus, the release was slower and the effect of pH was weakened. Hence, the release of curcumin from Cur-CMs was dependent upon both curcumin diffusion and chitosan degradation. In contrast, its release from Cur-PS-CMs was mainly controlled by diffusion. Considering the structure of Cur-PS-CMs, in which curcumin was complexed with phospholipids first and then loaded into chitosan microspheres as verified by DSC and FTIR, it could be concluded that the drug was released in two steps: (1) phytosomes were released from the microspheres; and (2) curcumin was released from phytosomes. This theory is in accordance with the studies of Feng et al. (2004), Stenekes et al. (2000) and Machluf et al. (1996), which all demonstrated that when liposomes are encapsulated in biodegradable microspheres, the systems first release intact liposomes before the drug is released. The attractive force between negatively charged PSs and positively charged chitosan

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Fig. 5. In vitro release of curcumin from chitosan microspheres (Values reported are the mean ± S.E.M.; n = 3). Table 1 Pharmacokinetic parameters of different curcumin formulations. Formulation

Cmax (ng/mL)

Natural curcumin Cur-PSs Cur-CMs Cur-PS-CMs

86.55 142.61 146.59 359.67

± ± ± ±

9.55 15.34* , a 16.31* , a 39.25* , a ; * , b ; * , c

Tmax (h) 0.60 1.20 1.40 2.00

± ± ± ±

0.13 0.27 0.22 0.00

AUC0–∞ (ng/mL h) 158.03 442.53 572.08 1184.21

± ± ± ±

18.05 91.89* , a 27.86* , a , # , b 221.07* , a ; * , b ; * , c

T1/2 (h) 1.21 1.73 2.34 3.16

± ± ± ±

0.23 0.38 0.29 0.50

Values are reported as mean ± S.E.M. (n = 5). Cmax : maximum concentration. Tmax : time to reach peak concentration. AUC: area under the plasma concentration–time curve from 0 h to ∞. T1/2 : half-life. a vs. natural curcumin. b vs. Cur-PSs. c vs. Cur-CMs. * p < 0.001. # p < 0.05.

played an important role in their combination, thereby affecting the releasing mechanism of chitosan microspheres. 3.5. Pharmacokinetic study We also investigated the feasibility of using Cur-PS-CMs as more efficient carriers to improve the bioavailability of curcumin. We compared relevant pharmacokinetic parameters after oral administration of natural curcumin, Cur-PSs, Cur-CMs, and Cur-PS-CMs at a curcumin concentration of 100 mg/kg body weight of rats. The maximum concentration (Cmax ) was detected in the blood at 30 min post-oral feeding of curcumin. Additionally, curcumin could not be detected beyond 6 h (Fig. 6). When Cur-PSs or Cur-CMs were given, Cmax was reached within 1.5 h and curcumin was undetectable at 8 or 10 h, respectively. Cur-PS-CMs exhibited Cmax at 2 h and showed

a sustained release of curcumin over 12 h post-feeding. The half-life (T1/2 ) of Cur-PS-CMs was 82.66% longer than that of Cur-PSs and 35.04% longer than that of Cur-CMs. The pharmacokinetic parameters showed that the absorption of curcumin improved significantly (Table 1). For Cur-PSs, Cur-CMs, and Cur-PS-CMs, Cmax was 142.61, 146.59, and 359.67 ng/mL, respectively, compared with the Cmax of 86.55 ng/mL exhibited by natural curcumin. The AUC0-∞ of Cur-PSs, Cur-CMs, and Cur-PS-CMs were 2.80, 3.62, and 7.49 times as much as natural curcumin, respectively. These results demonstrated that Cur-PS-CMs had a better sustained-release profile and better promoted oral absorption of curcumin than Cur-PSs or Cur-CMs. Thus, we conclude that a PS-CM delivery system, that is, a carrier combining chitosan microspheres and phytosomes, is a more favorable option for oral administration of curcumin than PSs and CMs. 4. Conclusion In our study, we successfully developed a novel drug delivery system, PS-CM, which was based on the combination of polymerbased and lipid-based controlled-release systems. Cur-PSs were effectively loaded in CMs by a simple method. In vitro drug release profiles indicated slow and sustained release of curcumin from PS-CMs. The PS-CMs significantly prevented degradation of bound curcumin in rat plasma and prompted absorption of curcumin compared with natural curcumin, Cur-PSs, and Cur-CMs. These preliminary studies show that PS-CMs can be promising vehicles for hydrophobic and rapidly eliminated compounds like curcumin, making them suitable for oral applications. Acknowledgments

Fig. 6. In vivo plasma concentration vs. time of different curcumin formulations. All values reported are the mean ± S.E.M. (n = 5).

This work was supported by the Projects of Chongqing Science & Technology (CSTC, 2011jjA0324), the Projects of Chinese

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