http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.924167
RESEARCH ARTICLE
Preparation and in vitro/in vivo evaluation of resveratrol-loaded carboxymethyl chitosan nanoparticles Yuangang Zu, Yin Zhang, Weiguo Wang, Xiuhua Zhao, Xue Han, Kunlun Wang, and Yunlong Ge
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Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang, China
Abstract
Keywords
Resveratrol (RES) is natural polyphenol with a strong biological activity, but its disadvantages, such as poor water solubility, susceptibility to oxidative decomposition and rapid metabolism in the body, which substantially restricts in vivo bioavailability, need to be resolved. This study used carboxymethyl chitosan (CMCS) as a drug carrier and utilized emulsion cross-linking to prepare RES-loaded CMCS nanoparticles (RES-CMCSNPs). A single-factor experiment was performed to optimize the preparation of these particles; in vitro and in vivo characteristics were evaluated. Spherical RES-CMCSNPs were prepared under optimal conditions, in which average particle size, potential, drug loading and encapsulation efficiency were (155.3 ± 15.2) nm, (10.28 ± 6.4) mV, (5.1 ± 0.8)% and (44.5 ± 2.2)%, respectively. FTIR, DSC and XRD showed that RES molecules were wrapped in the nanoparticles. In vitro DPPH radical scavenging abilities showed RES-CMCSNPs were better than RES raw powder. The nanoparticles improved the solubility of RES, thereby greatly improving the antioxidant activity of the drug. In vitro release experiments of RES and RES-CMCSNPs by simulating the human gastrointestinal tract were performed, in which RES-CMCSNPs rendered better releasing effects than raw RES. Raw RES and RES-CMCSNPs results were in line with those obtained for the single-chamber model for pharmacokinetic studies in rats. Compared with the bulk drugs, the RES-CMCSNPs exhibited increased in vivo absorption, prolonged duration of action and increased relative bioavailability by 3.516 times more than those of the raw RES. In addition, the residual chloroform is less than the ICH limit for class 2 solvents.
Antioxidant, bioavailability, carboxymethyl chitosan, nanoparticles, resveratrol
Introduction Resveratrol (RES) is a natural substance and an antioxidant produced by plants to protect against environmental damage. RES is mainly derived from Polygonum cuspidatum, grapes and peanuts. In-depth studies have confirmed that this substance has a strong biological activity that yields antitumor (Wu et al., 2004), antioxidative (Meyer et al., 1997), anti-bacterial and anti-inflammatory effects; RES also provides protection against cardiovascular and hepatic diseases, and participates in immune regulation (Pace-Asciak et al., 1995). RES is a non-flavonoid polyphenol with the chemical name 3,4,5-hydroxy-1,2-stilbene and molecular formula C14H12O3 (Figure 1). RES has many beneficial physiological activities in the human body; however, this substance has low water solubility, is susceptible to oxidative decomposition and has rapid metabolism and clearance rates in vivo, which substantially influences its absorption in the body and its bioavailability, as well as limits its beneficial effects (Baur & Sinclair, 2006). Pharmacokinetic studies have shown that the Address for correspondence: Xiuhua Zhao, Associate Professor, Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, Heilongjiang, China. Tel: +86 451 82191517. Fax: +86 451 82102082. E-mail: [email protected]
History Received 26 March 2014 Revised 10 May 2014 Accepted 11 May 2014
oral bioavailability of RES is almost zero (Wenzel & Somoza, 2005). Therefore, increasing the water solubility of RES, enhancing the impact of its oral absorption and improving its bioavailability are problems that need to be addressed. Researchers have been increasingly concerned about new solubilization technologies, such as synthetic water-soluble prodrug, cyclodextrin inclusion, nanotechnology, microemulsions, liposomes and mixed micelles to improve the efficacy of poorly soluble drugs. Nanoparticle drug systems use nanoparticles as drug carriers made from natural or synthetic polymer materials. In these systems, the drug molecules covalently couple to nanoparticles by chemical methods or are wrapped and adsorbed by physical methods. Given the small size of nanoparticles, crossing biological membranes is easy, thereby effectively improving the oral bioavailability of poorly soluble drugs. This system has become important in pharmaceutical research with the development of nanotechnology. Studies have shown that the nanoparticle drug system has significant advantages, including increasing the solubility of drugs in the body, thereby enhancing absorption and improving in vivo bioavailability. This system also prolongs the effect of the drug, which achieves the slow-release effect. Drugs are controlled into specific target organs or cells upon using this system, which reduces drug side effects
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used to detect particle size, drug loading, encapsulation efficiency, physical, chemical and release characteristics through antioxidative and bioavailability studies. These technologies were also used to obtain excellent stability, high bioavailability and more desirable effects of nanoformulated oral RES and the corresponding preparation processes and quality control standards.
Materials and methods
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Figure 1. The molecular formulation of resveratrol.
(Kreuter, 2001). This system improves drug stability and prevents the drug from being degraded before reaching the lesion (Takeuchi et al., 2001). Drug absorption is also enhanced, namely, nanoparticles allow the drug to penetrate easily through biological membranes and pass through the cell gap and the blood–brain barrier to reach the diseased tissue. This system also uses carrier materials that are biodegradable and non-toxic (or less toxic). The nanoparticle drug system can be divided according to preparation process and materials, namely, nanoparticle suspensions (Elzoghby et al., 2012), solid lipid nanoparticles (Wissing et al., 2004), nanoemulsion (Tahara et al., 2011), nano-liposomes (Xiong et al., 2011) and nano-micelles (Wang et al., 2009). Biological macromolecular materials as nano-drug carriers have received increasing attention because of their renewable resources. These materials are biological adhesives having no toxicity and good biocompatibility, thereby promoting ease of degradation in vivo. Synthetic polymers and natural polymer materials are used to prepare nano-drug carriers. Examples of synthetic polymers include polyester-based aliphatic polyesters, aromatic polyesters, polycarbonates, polyanhydrides and polyamides (Panagi et al., 2001; Watnasirichaikul et al., 2002). Synthetic macromolecular materials can synthesize the desired product according to the specific need and can be modified to control the characteristics, yielding a product that is highly purified, non-toxic and readily degradable in vivo. Natural polymer materials are also commonly used as nano-drug carriers, which are classified as proteins and polysaccharides. Proteins include gelatin, serum and plant proteins; polysaccharides include cellulose, starch, sodium alginate, chitosan and cyclodextrin. Natural flora and fauna are the main sources of these materials, indicating abundant renewable resources. These materials have good biocompatibility, and can be degraded enzymatically in vivo. Natural polymer materials are ruled out in vitro from non-toxic small molecules (Horan et al., 2005) that also possess molecular chains with hydroxyl, amino and carboxyl groups used as chemical modification sites, thereby exhibiting broad applications as drug carrier materials. Carboxymethyl chitosan (CMCS), which is produced from the reaction of chitosan with chloroacetic acid in an alkaline condition, is a biological macromolecule material widely applied as a pharmaceutical carrier material (Fu et al., 2011). This substance is a bioadhesive that has good water solubility, biocompatibility, biodegradability and can promote permeability (Dong et al., 2010). In this study, CMCS was used as the drug carrier to carry RES by emulsion cross-linking. Single-factor experiments were used to optimize the preparation of nanoparticles. Physical, chemical and in vitro detection technologies were
Materials Resveratrol (purity ¼ 98%) was obtained from Shanxi Sen Fo Gao Ke Co., Ltd. (Xi’an, Shanxi, PR China). CMCS (molecular weight ¼ 14.2 104 Da) was sourced from Zhejiang Ao Xing Biotechnology Co., Ltd. (Yuhuan, Zhejiang, PR China). Tween-80, anhydrous calcium chloride, mannitol, glucose, lactose, alpha-cyclodextrin, beta-cyclodextrin and gammacyclodextrin were all obtained from Jiangsu Fengyuan Biotechnology Co., Ltd. (Suqian, Jiangsu, PR China). DPPH was from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Male rats were provided by Harbin Medical University (Harbin, Heilongjiang, PR China). Methanol, ethanol, chloroform and other reagents were of analytical grade. Preparation and optimization of RES-CMCSNPs by single-factor optimization Based on related literature and pre-experimental results, the key factors affecting the preparation of nanoparticles were CMCS concentration (in mg/ml), water:chloroform (V/V), Tween-80 ratio, homogenization time, number of highpressure homogenizers, high-pressure homogenization pressure and CaCl2 concentration. Emulsion cross-linking method was carried out stepwise using a single-factor optimization in each step to obtain the optimal conditions in preparing RESCMCSNPs. Carboxymethyl chitosan was completely dissolved in ultrapure water, and the impurities were removed by filtration. Appropriate amounts of Tween-80 were added after mixing into the CMCS solution, and a certain amount of chloroform was added dropwise to the solution by homogenization at a speed of 10 000 rpm. CMCS formed colostrum. Factors affecting emulsification of CMCSNPs included CMCS concentration (in mg/ml), water to chloroform ratio, Tween-80 ratio and homogenization time. Nanoparticle size was the indicator of the inspection. Other factors were held constant upon examination of one of the factors. CMCS concentrations varied from 2 to 6 mg/ml, with 1 mg/ml increment; water to chloroform ratios had values of 5:1, 10:1, 15:1, 20:1 and 25:1. Tween-80 ratios varied from 1/1000 to 5/1000, with an increment of 1/1000. Homogenization times varied from 1 to 9 min, with 2-min increments. Homogeneity implies that materials were refined and uniformly mixed in the homogenizing valve and in the emulsified solution after the high-pressure homogenizer, thereby obtaining more uniform, more stable and smaller nanoparticles. The main factors affecting high-pressure homogenization were homogenization pressure. Nanoparticle size was the indicator of the inspection. Homogenization pressure varied from 400 to 1200 bars, with increments of 200 bars.
Resveratrol-loaded carboxymethyl chitosan nanoparticles
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DOI: 10.3109/10717544.2014.924167
The number of heterogeneous particles varied from 3 to 15 times, with increments of 3 times. After completion of the emulsified portion, we used anhydrous calcium chloride as the cross-linking agent for ionic cross-linking. CMCS is an amphoteric electrolyte that has a negative charge under alkaline conditions. The weak acid anionic group (COO) and Ca2+ ions in inter- or intramolecular cross-linking reaction could occur to obtain nanoparticles. Added CaCl2 quality can directly affect the particle size of the nanoparticles, potential, entrapment efficiency and drug-loading amount, thereby classifying CaCl2 concentration as a factor in obtaining optimal CaCl2 qualities through single-factor experiment. Tween-80 was added to the CMCS solution and RES was sufficiently dissolved in chloroform. Chloroform was added dropwise to the CMCS solution under stirring of a homogenizer. Good emulsion was obtained after the homogenate and the highpressure homogenizer were used. CaCl2 concentrations from 1 to 6 mg/ml were selected, with a 1 mg/ml increment. CaCl2 solution was added dropwise to the emulsion until completion of the dropping. Following the completion of cross-linking, we detected the particle size and potential of each sample. Chloroform was removed using a rotary evaporator in vacuum, and was centrifuged at 10 000 rpm for 30 min. The nanoparticles were separated, the precipitate and the supernatant were collected and the precipitate was washed twice with ultrapure water. The supernatant was used to determine encapsulation efficiency and drug loading. RES-CMCSNPs were freeze-dried at 50 C for 48 h. In this process, 50% (mannitol percentage of RES-CMCS quality) mannitol was selected as lyophilized protective agent of these particles.
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Surface morphology of nanoparticles The morphology of raw RES powder, raw CMCS powder, freeze-dried RES-CMCSNPs powder and added lyoprotectant freeze–dried RES-CMCSNPs powder was ascertained through scanning electron microscopy (SEM, S4800, Hitachi, Ltd., Tokyo, Japan). After the high-pressure homogenizer was used, morphological changes of the nanoparticles were analyzed via electron microscopy (BH-2, Olympus Corporation, Tokyo, Japan). Chemistry characterization of nanoparticles The molecular structures of RES raw powder and nanoparticles were examined with Fourier transform infrared (FTIR) spectroscopy using a MAGNA-IR560 E.S.P (Nicolet Pittsburgh, PA, USA) instrument. The spectra were obtained in transmission mode at room temperature in 4000–400 cm1 range at a resolution of 2 cm1. Physical status of RES in RES-CMCSNPs An X-ray diffractometer (Philips, Xpert-Pro, The Netherlands) was used to determine the physical status of paclitaxel in the nanoparticles. The diffraction angle (2y) was recorded from 3 to 80 with a scanning speed of 5 /min. The samples were irradiated using a Cu target tube at 30 mA current and 40 kV voltage. Thermal analysis was performed with differential scanning calorimetry (DSC, TA Instruments, model DSC 204, Woodland, CA, USA). All thermal analyses were performed for 5.0 mg samples at a heating rate of 10 C/min and a temperature range of 20–300 C.
Characterization of RES-CMCSNPs
GC analysis
Size, zeta potential, drug encapsulation and loading efficiency
The residual chloroform in the RES-CMCSNPs were analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with DB-WAX polyethylene glycol capillary column (30.0 m 250 mm 0.25 mm, nominal) equipped with a G1540N-210 FID detector (Agilent Technologies, San Jose, CA, USA). Peaks areas were used for obtaining quantitative data. The conditions of GC analysis of chromatograph were as follows: oven temperature was maintained at 40 C for 5 min initially, and then raised at the rate of 10 C/min to 200 C, which was maintained for 3 min at last. The injector and the detector temperatures were set 200 C and 250 C, respectively. Nitrogen was used as carrier gas at a flow rate of 25 ml/ min, and 5 ml samples were injected manually in the split mode with a split ratio 25:1. Hydrogen gas and air flow rate were 30 and 400 ml/min.
The mean particle size and electric potential of the obtained nanoparticles were analyzed by dynamic light scattering equipment (ZetaPALS, Brookhaven Instruments, Long Island, NY, USA). The samples were prepared by dissolving the powdered nanoparticles with deionized water, and the suspension was sonicated for a while. Each experimental preparation was executed in triplicate, and data were obtained from the average of several measurements. Free RES in the supernatant was tested by high-performance liquid chromatography (HPLC) using a Diamonsil C18 column (250 mm 4.6 mm, 5 mm; Dikma Technologies, Beijing, China). An HPLC system (Waters Corporation, Milford, MA, USA), consisting of a Waters 600 Controller equipped with a Waters 717 plus autosampler, and a Waters 2487 UV detector were used. Ten microliters of the sample was injected into a C18 column at 25 C, with a 40:60 water (0.25% acetic acid)/ methanol mobile phase. The elution flow rate was 1 ml/min, and detection was accomplished at 306 nm. DEE and DLE were calculated using the following previously reported equations: DEE¼
ResðtotalÞ ResðfreeÞ 100% ResðtotalÞ
ð1Þ
DLE¼
ResðtotalÞ ResðfreeÞ 100% RES CMCSNPs
ð2Þ
DPPH radical scavenging activity assay of RES-CMCSNPs In this assay, 25.76 mg DPPH powder was added to 50% ethanol solution to yield a concentration of 6.5 104 mol/l of DPPH stock solution. Three samples were employed: the RES raw powder group, the RES-CMCSNPs group containing the same quality of RES and the CMCSNPs group with the same quality of that in RES-CMCSNPs. Each group used PBS (4 mM, pH ¼ 7.4) to configure different concentration
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gradients into five samples (0.0625, 0.125, 0.25, 0.5 and 1 mg/ ml). Up to 3.5 ml of DPPH radical solution was mixed with 0.5 ml of three sample groups at various concentrations. Absorbance at 517 nm was measured after 60 min in an incubator at 37 C, in which each absorbance value was measured in triplicate. PBS was employed as blank control. DPPH radical scavenging ability was calculated as Scavenging ability% ¼
A0 As 100 As
ð3Þ
where A0 is the absorbance of the control and AS is the absorbance of the tested sample.
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In vitro RES release from RES-CMCSNPs The condition of in vitro oral drug release was at 37 C, thereby using artificial gastric juice and intestinal fluid to simulate the human body environment. One thousand milliliters of artificial gastric juice and intestinal juice were used according to the Pharmacopoeia records. RES was not conducive for detection because of insolubility in water. We added 1 ml of Tween-80 into the artificial gastric and intestinal fluids to allow more uniform dispersion dissolution of RES. Five milligrams of RES raw powder and lyophilized RESCMCSNPs powder containing the same quality of RES were dissolved in 10 ml of artificial gastric and intestinal fluids, and were placed in dialysis bags that were, respectively, suspended in two 250-ml beakers containing 200 ml fluids. The beakers were placed on magnetic stirrers and reacted at a constant temperature of 37 C. The speed was maintained at 50 rpm according to the in vivo rate of gastric peristalsis. At predetermined time intervals, 3 ml dialysate was withdrawn for detection, and equal volumes of fresh artificial gastric and intestinal fluids were added each time. The concentration of RES was determined by highperformance liquid chromatography. Liquid detection conditions corresponded with previously described conditions. Cumulative release percentage was calculated with the following formula: Ci0 ¼ C1 0 ¼ Ciþ1 Ciþ1 i P
Qi ¼
i¼1
ð4Þ
ðV Vi Þ Ci V
ð5Þ
Ci0 V M
100%
ð6Þ
where Ci is the RES concentration of each sample withdrawn at predetermined time intervals, Ci0 is the increase in RES concentration during each time interval, V is the volume of the release buffer, Vi is the volume of each withdrawn sample, M represents the RES loaded in the RES-CMCSNPs and Qi represents the accumulative release percentage at a predetermined time point. Drug release studies were conducted in triplicate for each sample. In vivo bioavailability studies of RES-CMCSNPs in rats Six healthy male SD rats were used and divided into two groups according to similarity in weight. Three parallel tests
were performed for three rats of each group. Rats were fasted for 12 h before administration. RES raw powder (control group) and RES-CMCSNPs (experimental group) were administered orally to the rats, with 50 mg/kg dosage (calculated by RES). After administration, 1 ml of blood was collected from the fundus after administration. Sampling times for the control group were 0.5, l, 2, 3, 4, 5, 6, 8, 12 and 16 h, and that for the experimental group were 0.5, l, 2, 3, 4, 5, 6, 8, 12, 24 and 48 h. Blood was placed in a heparinized centrifuge tube, and was centrifuged for 5 min at 3500 rpm. Upper plasma was taken following the centrifugation. One hundred microliters of plasma was placed in a 1.5-ml Eppendorf tube, followed by the addition of 300 ml methanol. After vortex mixing for 3 min and centrifugation for 10 min at 10 000 rpm, the upper organic layer was drawn to dry with nitrogen in a water bath at 40 C. The residue was dissolved with 100 ml methanol, and 20 ml of the supernatant was taken for injection. In this process, the methanol:water (0.25% acetic acid) mobile phase was 60:40 (v/v), the flow rate was 1 ml/min, the column temperature was 30 C, the detection wavelength was 306 nm and the injection volume was 20 ml.
Results and discussion Preparation and optimization of RES-CMCSNPs Emulsification of CMCSNPs Figure 2(a) shows that the concentration of CMCS had a marked effect on the particle size of CMCSNPs upon fixing three other factors. The particle size of CMCSNPs increased with the concentration of CMCS, which was attributed to the increase in viscosity upon the increase in CMCS concentration, which easily agglomerated the particles and resulted in increased particle size. If the particle size of CMCSNPs was too small, the nanoparticles were more difficult to separate. Moreover, absorption would be affected if the particle size was too large. The drug could not be fully contacted if the concentration of CMCS was relatively low, thereby affecting the drug loading. Thus, we chose CMCS concentration of 5 mg/ml as the optimization condition. Figure 2(b) shows that the volume ratio of water and chloroform likewise significantly affected the particle size of CMCSNPs. The particle size of CMCSNPs increased with volume ratio of water and chloroform, whereas three other factors were held constant. Based on particle size, the 10:1 volume ratio of water and chloroform was determined as the optimal condition. Figure 2(c) shows that as the three other factors were held constant, the ratio of Tween-80 and water had no significant effects on the particle size of CMCSNPs, which was 150 nm. As additives to drugs, the proportion of Tween80 would affect the human body. Taking safety into account, we chose 1/1000 as the optimization criteria. Figure 2(d) shows that the homogenization time of CMCSNPs also affected particle size, with the three other factors held constant. From 1 to 3 min, the particle size decreased with the increase of homogenization time because a short time led to an uneven emulsification. Some particles agglomerated together, resulting in a slightly larger particle size. The particle size decreased with the increase of the
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DOI: 10.3109/10717544.2014.924167
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Figure 2. Impact trend graph of: (a) CMCS concentration, (b) the volume ratio of water and chloroform, (c) the ratio of Tween-80 and water, (d) homogenized time, (e) the pressure of high-pressure homogenization and (f) the time of high-pressure homogenization to CMCSNPs size.
homogenization time. In the following 3–9 min, the particle size of CMCSNPs increased with the homogenate time, because the emulsification time was longer and mechanical stirring produced mechanical energy that increased the energy of the. To reduce this energy, the particles should be closer to each other, thereby reducing the specific surface area and increasing the particle size. Based on changes in particle size, we selected 5 min as the appropriate time of emulsification. Figure 2(e) shows that pressure affects the size of CMCSNPs during high-pressure homogenization. When the number of homogenization was fixed, increasing the pressure initially reduced the size of the nanoparticles because the increased homogenization pressure produced a large mechanical force. The particle size was too large or uneven, and gradually became smaller and more uniform after homogenization. When the pressure exceeded 600 bar, the particle size increased with pressure.
Figure 2(f) shows that when the homogenization pressure was fixed, the particle size decreased with increasing number of homogeneous particles because this increase lengthened the mechanical action time. The particles reduced after homogenization. When the number of homogeneous particles constantly increased, the particle size also increased High-pressure homogenization resulted in reduced and uniform particle size. However, the size of nanoparticles increased when the pressure and number of homogenization continued to increase up to a certain value because the pressure was high or homogenization was performed too many times. Aggregation between the particles repeatedly increased the average particle size. The limited emulsifier could not be effectively adsorbed to the particle surfaces, thereby reducing the emulsification, aggregating the droplets, increasing the particle size and causing instability. Ultimately, we chose 600 bar as the pressure of high-pressure
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Figure 3. Impact trend graph of CaCl2 concentration to size, zeta potential, drug loading and encapsulation efficiency of RES-CMCSNPs. (a) Size, (b) zeta potential, (c) drug loading and (d) encapsulation efficiency.
homogenization, and six times as the optimal number of homogenization times. Ionic cross-linking Figure 3 shows that at constant volume, CaCl2 concentration had varying effects on size, zeta potential, drug loading and encapsulation efficiency of RES-CMCSNPs. Figure 3(a) reveals that particle size increased with CaCl2 concentration because the increase of Ca2+ had ionic cross-linking with negatively charged groups (COO) on CMCS, thereby allowing more CMCS molecules to combine and yield a larger particle size. The emulsions were negatively charged prior to the addition of CaCl2. When the increased Ca2+ cross-linked together with negative charges, the negative charges of the emulsion decreased. Figure 3(b) shows that the zeta potential of RES-CMCSNPs increased with CaCl2 concentration. Figure 3(c) and (d) shows that drug loading and encapsulation efficiency increased with CaCl2 concentration. When this concentration reached 5 mg/ml, the drug loading and encapsulation efficiency were, respectively, 5.1% and 44.3% of the maximum. Further increasing this concentration to 6 mg/ml stopped the increase of both drug loading and encapsulation efficiency. When the CaCl2 concentration was 5 mg/ml, Ca2+ and CMCS quality reached a suitable ratio, which achieved an optimal state for the Ca2+ and CMCS cross-linking. At this point, the particle size and potential were the most stable and appropriate, in which the drug
loading and encapsulation efficiency reached maximum values. Therefore, we selected the CaCl2 concentration of 5 mg/ml as the optimum condition for ionic cross-linking. Characterization of RES-CMCSNPs Size, zeta potential, drug encapsulation and loading efficiency According to the optimal formulation, validation tests were carried out in triplicate. Results showed that the average nanoparticle size was (155.3 ± 15.2) nm, the potential was (10.28 ± 6.4) mV, drug loading was (5.1 ± 0.8)% and encapsulation efficiency was (44.5 ± 2.2)%. The batch reproducibility was good. Surface morphology of nanoparticles An optical microscope was used to observe and compare the morphologies of RES-CMCSNPs. Figure 4(a) shows the morphology of RES-CMCSNPs without homogenization in different multiples. The particle sizes were relatively large and uneven, and a two-layer structure could be observed. The outer layer should be aqueous phase with CMCS, and the inner is the oil phase containing RES, thereby successfully proving the initial emulsion. Figure 4(b) shows the morphology of RES-CMCSNPs after homogenization in different multiples. Compared with that shown in Figure 4(a), the particle size was significantly smaller and became more uniform after high-pressure homogenization.
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DOI: 10.3109/10717544.2014.924167
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Figure 4. Different multiples morphology of RES-CMCSNPs: (a) without homogenization (10 10) and (b) after homogenization (10 10).
Figure 5. Scanning electron micrograph: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 5 shows the scanning electron micrograph of RES raw powder, CMCS, RES-CMCSNPs and RES-CMCSNPs with mannitol. Figure 5(a) and (b) shows that the RES raw powder exhibited a needle crystal structure, and raw CMCS powder was in amorphous powder state. Figure 5(c) shows
that RES-CMCSNPs were spherical and have relatively uniform sizes. Figure 5(d) shows that mannitol as the freezing-drying protective agent has a significant function in protecting RES-CMCSNPs as the nanoparticles were embedded.
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Figure 6. Infrared spectrogram: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 7. XRD pattern: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs, (d) RES-CMCSNPs with mannitol and (e) mannitol.
Chemistry characterization of nanoparticles
XRD pattern of RES-CMCSNPs with mannitol, in which peaks appeared at 6.5 , 16.2 and 19.0 , indicating the crystal structure of mannitol. Figure 7(e) shows the XRD pattern of mannitol, in which peaks appeared at 14.8 , 19.0 and 23.5 , which indicate the crystal structure of mannitol. Figure 8(a) represents the DSC thermogram of RES raw powder. An obvious endothermic peak existed when the temperature increased to 267.82 C, which was very close to the melting point of RES, indicating that RES crystals have melted. Figure 8(b) shows that CMCS has no obvious endothermic or exothermic peak, proving that CMCS has amorphous state structures. Figure 8(c) shows the DSC thermogram of RES-CMCSNPs, which is very similar to that of CMCS. No apparent melting processes were observed, which revealed that RES-CMCSNPs have an amorphous state structure. Figure 8(d) shows the DSC thermogram of RESCMCSNPs with mannitol. A crystal fusion endothermic peak exists when the temperature has reached 164.4 C, indicating the crystal structure of mannitol.
Figure 6(a) shows the infrared spectrogram of the RES raw powder. A phenolic hydroxyl group absorption peak at 3249 cm1 and benzene ring absorption peaks at 1622, 1540 and 1506 cm1 exist. Figure 6(b) shows the infrared spectrogram of CMCS, in which a hydroxyl group absorption peak at 3424 cm1 exists. Figure 6(c) shows the infrared spectrum of RES-CMCSNPs. The characteristic absorption peak appeared at the same position, but the characteristic absorption peak of RES did not appear upon comparison with Figure 7(b), proving that RES had been completely wrapped in CMCS. Figure 6(d) shows the infrared spectra of RES-CMCSNPs with mannitol. In addition to the characteristic absorption peaks of CMCS, the characteristic peaks of mannitol were also markedly observed. Physical status of RES in RES-CMCSNPs Figure 7(a) shows the XRD pattern of RES raw powder. Multiple characteristic peaks for RES existed because of the crystalline structure. Figure 8(b) shows the XRD pattern of CMCS, in which characteristic peaks from 5 to 50 were not observed, indicating that CMCS has amorphous structures. Figure 7(c) shows the XRD pattern of RES-CMCSNPs, which is similar to that in Figure 7(b), implying that RES is almost completely encapsulated in CMCS. Figure 7(d) shows the
Solvent residue analysis The problem of solvent residues is also under consideration in pharmaceutical products. In this article, emulsion crosslinking method was carried out to paper RES-CMCSNPs. Figure 9(a) and (b) shows the results of chloroform residue
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DOI: 10.3109/10717544.2014.924167
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Figure 10. DPPH radical scavenging rate. (a) RES raw powder, (b) RES-CMCSNPs with CMCSNPs as control, (c) RES-CMCSNPs and (d) CMCSNPs.
DPPH radical scavenging activity assay of RES-CMCSNPs
Figure 8. DSC thermogram: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 10(a), (c) and (d) shows the DPPH radical scavenging ability of RES raw powder, RES-CMCSNPs and CMCSNPs. Figure 10(b) is the practically measured result of the DPPH radical scavenging ability of RES-CMCSNPs with CMCSNPs as control. As seen from Figure 10(c), CMCSNPS itself have little antioxidant activity. In the range of 62.5–1000 mg/ml, the clearance rate also gradually increased with the concentration of RES. When the concentration was 1000 mg/ml, the clearance rate reached almost 70%. The DPPH radical scavenging capacities of RES-CMCSNPs were considerably better than those of the RES raw powder. When the samples reacted with the DPPH radical, 50% ethanol solution was introduced to improve the solubility of RES. Thus, minimal difference existed between low-concentration samples of RES raw powder and RES-CMCSNPs. When the concentration was 1000 mg/ml, the DPPH radical scavenging rate of RESCMCSNPs was slightly higher than that of RES raw powder. DPPH radical scavenging experiment showed that the antioxidant activity of RES substantially improved, which possible reason is that RES-CMCSNPs improved the watersolubility of RES raw powder. In vitro RES release from RES-CMCSNPs
Figure 9. Gas chromatograms of samples: (a) chloroform standard solution and (b) RES-CMCSNPs.
using the GC method. From the chromatograms of chloroform standard solution, a regression equation between peak area (Y) and ethanol concentration (x) can be fitted as Y ¼ 127.1015 x + 0.6195 (R2 ¼ 0.9998). The linear range of chloroform was 0.0015625–0.1 mg/ml. According to the regression equation, the residual chloroform content in RES-CMCSNPs is 60 ppm. Since the ICH limit for chloroform in class 2 solvents is 60 ppm or 0.006%, the RES-CMCSNPs met ICH requirements and are suitable for pharmaceutical use.
Figure 11(a) and (b), respectively, represents the release profiles of RES raw powder and RES-CMCSNPs in a simulated gastric fluid. Figure 11(a) shows that RES raw powder is quickly released within the first 2 h when the cumulative release percentage has reached 26.9% because of a small amount of the raw powder that is completely dissolved and has a quick release process. At a later time, RES raw powder is slowly released until 68 h when the cumulative release percentage has reached only 65.9%. However, the RES raw powder was inadequately dissolved in the dialysis bag because of the poor solubility of RES raw powder in water. Upon the release of RES from the dialysis bag, the degree of saturation changed. Undissolved RES gradually dissolved in the dialysis bag and was continuously released. Thus, the
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Table 1. Pharmacokinetic parameters after gavage. Atrioventricular parameters
RES
RES-CMCSNPs
A (mg/ml) Ke (1/h) Ka (1/h) t1/2 (ka) (h) t1/2 (ke) (h) Tmax (h) Cmax (mg/ml) AUC ((mg/ml)h) CL/f(s) (mg/h/(mg/ml)) V/f(c) ((mg)/(mg/ml))
3.95 0.13 0.62 1.12 5.44 3.21 2.00 24.60 0.41 3.19
3.19 0.03 0.39 1.76 20.55 6.83 2.32 86.50 0.12 3.43
Figure 11. Release profile in simulated gastric fluid. (a) RES raw powder and (b) RES-CMCSNPs.
Figure 13. Concentration–time curve. (a) RES raw powder and (b) RES-CMCSNPs.
Figure 12. Release profile in simulated intestinal fluid. (a) RES raw powder and (b) RES-CMCSNPs.
cumulative release percentage was initially low but gradually increased. Figure 11(b) shows the RES-CMCSNPs release profile divided into two phases: burst release and sustained release periods. The first 1 h was burst release period because of a small amount of drug loosely bound in the surface of nanoparticles, causing rapid drug release. Thereafter, during the sustained period, RES wrapping in nanoparticles became more difficult through microporous diffusion, and the release depends on the degradation of CMCS. At 44 h, the cumulative release percentage reached a maximum of 82.8%, indicating that the RES-CMCSNPs were almost completely released. The cumulative release percentage reached 81.3% in 68 h, and was higher than that of RES raw powder. Figure 12(a) and (b), respectively, represents the release profiles of RES raw powder and RES-CMCSNPs in simulated intestinal fluid, which were similar to the release in simulated gastric fluid. RES raw powder had a burst release process in the first 2 h, and the cumulative release percentage reached 33.7%. When released at 68 h, the cumulative release percentage reached 64.8%. Slow release was observed, and the dialysis bag still contained the undissolved RES raw
powder. By contrast, the cumulative release percentage of RES-CMCSNPs reached 34.1% within 1 h. When time reached 44 h, the cumulative release percentage reached a maximum of 80.5%, indicating that the RES-CMCSNPs were almost completely released. The cumulative release percentage reached 79.2% in 68 h, and was higher than that of the RES raw powder. The drug release profile was analyzed with OriginPro 8.5.1 software (Northampton, MA, USA). In the simulated gastric juice, release characteristics of the RES raw powder agreed withx the Higuchi equation y ¼ 30:93 x e¼17:68 36:84e1:57 þ 65:12 (R2 ¼ 0.990). The same trend was observed xfor the release curve of RES-CMCSNPs: x y ¼ 48:61e7:95 36:11e0:44 þ 80:90 (R2 ¼ 0.993). The release curves for RES raw powder and RES-CMCSNPs in the intestinal fluid also agree withxthe Higuchi equation, given x by y ¼ 27:85ex20:66 40:90ex1:01 þ 64:66 (R2 ¼ 0.995) and y ¼ 51:09e8:47 31:044e0:5 þ 80:66 (R2 ¼ 0.996). In vivo bioavailability studies of RES-CMCSNPs in rats The 3p87 software (Chinese Pharmacological Society, Beijing, PR China) was used to model fit the data, in which we obtained various kinetic parameters of RES raw powder and RES-CMCSNPs (Table 1). They were single-room models. The weighing coefficients were both 1/C/C. Figure 13 shows the plasma concentration–time curve.
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DOI: 10.3109/10717544.2014.924167
After oral administration, in vivo absorption and elimination of RES raw powder were rapid, in which t1/2 (ka) was 1.12 h, t1/2 (ke) was 5.44 h, peak time was 3.21 h, maximum plasma concentration was 2.00 mg/ml and the area under the concentration–time curve AUC was 24.60 (mg/ml)h. After oral administration of RES-CMCSNPs, half-life t1/2 (ka) extended to 1.76 h, elimination half-life t1/2 (ke) extended to 20.55 h, peak time reached 6.83 h, maximum plasma concentration increased to 2.32 mg/ml and the area under the concentration–time curve for AUC increased to 86.50 (mg/ ml)h, was 3.516 times that of RES. The relative bioavailability of RES-CMCSNPs was 3.516 times as large as RES. The maximum plasma concentration time of nanoparticles was later than that of the RES raw powder, which further proved that nanoparticles exhibited sustained release effect, thereby extending the drug peak time. The drug was wrapped in CMCS, which prevented the removal of the drug from the body, and induced the clearance rate to decrease and the elimination half-life to significantly increase. These phenomena extended the presence time of the drug in the body and contributed to the improvement of bioavailability. Comparing the concentration–time curve and the kinetic parameters, the bioavailability of nanoparticles in rats substantially improved. One reason may be the improvement of RES water solubility. Another reason may be that particle size was suitable for uptake by intestinal cells and CMCS as a drug carrier, endowing the nanoparticles with biological adhesion and promoting permeability advantages, thus extending the gastrointestinal residence time of RES and aiding in the absorption of drugs.
Conclusions This study attempts to improve the oral bioavailability of RES, and CMCS as a drug carrier. RES nanoparticles were prepared by emulsion cross-linking. Single-factor experiment was used to obtain the optimal conditions for nanoparticles. RES-CMCSNPs were spherical with uniform particle size distribution, and RES was completely wrapped in the nanoparticles. Oxidation in vitro experimental tests showed that the nanoparticles improved the water solubility of RES, thereby improving the antioxidant activity of the drug. In vitro release test showed the good release effect of RESCMCSNPs. In vivo bioavailability study of drugs showed that nanoparticles had better absorption in the body, in which the relative bioavailability was increased 3.516 times compared with the RES raw powder. Results indicated that the nanoparticle drug system promoted the absorption of RES, improved the raw powder which was initially insoluble in water and improved its oral bioavailability. In addition, the residual chloroform is less than the ICH limit for class 2 solvents. In summary, this article provides a theoretical and experimental basis for solving poor water solubility and low oral bioavailability of RES.
Resveratrol-loaded carboxymethyl chitosan nanoparticles
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Acknowledgements The authors are grateful for the precious comments and careful corrections made by anonymous reviewers.
Declaration of interest The authors would also like to acknowledge the financial support from the Fundamental Research Funds for the Central Universities (DL12EA01-02) and the National Natural Science Foundation of China (No. 21203018).
Reference Baur JA, Sinclair DA. (2006). Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov 5:493–506. Dong W, Han B, Feng Y, et al. (2010). Pharmacokinetics and biodegradation mechanisms of a versatile carboxymethyl derivative of chitosan in rats: in vivo and in vitro evaluation. Biomacromolecules 11:1527–33. Elzoghby AO, Samy WM, Elgindy NA. (2012). Albumin-based nanoparticles as potential controlled release drug delivery systems. J Control Rel 157:168–82. Fu D, Han B, Dong W, et al. (2011). Effects of carboxymethyl chitosan on the blood system of rats. Biochem Biophys Res Commun 408: 110–14. Horan RL, Antle K, Collette AL, et al. (2005). In vitro degradation of silk fibroin. Biomaterials 26:3385–93. Kreuter J. (2001). Nanoparticulate systems for brain delivery of drugs. Adv Drug Deliv Rev 47:65–81. Meyer AS, Yi O-S, Pearson DA, et al. (1997). Inhibition of human lowdensity lipoprotein oxidation in relation to composition of phenolic antioxidants in grapes (Vitis vinifera). J Agric Food Chem 45: 1638–43. Pace-Asciak CR, Hahn S, Diamandis EP, et al. (1995). The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta 235:207–19. Panagi Z, Beletsi A, Evangelatos G, et al. (2001). Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA-mPEG nanoparticles. Int J Pharm 221:143–52. Tahara K, Miyazaki Y, Kawashima Y, et al. (2011). Brain targeting with surface-modified poly(D,L-lactic-co-glycolic acid) nanoparticles delivered via carotid artery administration. Eur J Pharm Biopharm 77:84–8. Takeuchi H, Yamamoto H, Kawashima Y. (2001). Mucoadhesive nanoparticulate systems for peptide drug delivery. Adv Drug Deliv Rev 47:39–54. Wang ZY, Song J, Zhang DS. (2009). Nanosized As2O3/Fe2O3 complexes combined with magnetic fluid hyperthermia selectively target liver cancer cells. World J Gastroenterol 15:2995–3002. Watnasirichaikul S, Rades T, Tucker IG, Davies NM. (2002). Effects of formulation variables on characteristics of poly (ethylcyanoacrylate) nanocapsules prepared from w/o microemulsions. Int J Pharm 235: 237–46. Wenzel E, Somoza V. (2005). Metabolism and bioavailability of transresveratrol. Mol Nutr Food Res 49:472–81. Wissing SA, Kayser O, Muller RH. (2004). Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev 56:1257–72. Wu SL, Sun ZJ, Yu L, et al. (2004). Effect of resveratrol and in combination with 5-FU on murine liver cancer. World J Gastroenterol 10:3048–52. Xiong S, Yu B, Wu J, et al. (2011). Preparation, therapeutic efficacy and intratumoral localization of targeted daunorubicin liposomes conjugating folate-PEG-CHEMS. Biomed Pharmacother 65: 2–8.
RESEARCH ARTICLE
Preparation and in vitro/in vivo evaluation of resveratrol-loaded carboxymethyl chitosan nanoparticles Yuangang Zu, Yin Zhang, Weiguo Wang, Xiuhua Zhao, Xue Han, Kunlun Wang, and Yunlong Ge
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Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang, China
Abstract
Keywords
Resveratrol (RES) is natural polyphenol with a strong biological activity, but its disadvantages, such as poor water solubility, susceptibility to oxidative decomposition and rapid metabolism in the body, which substantially restricts in vivo bioavailability, need to be resolved. This study used carboxymethyl chitosan (CMCS) as a drug carrier and utilized emulsion cross-linking to prepare RES-loaded CMCS nanoparticles (RES-CMCSNPs). A single-factor experiment was performed to optimize the preparation of these particles; in vitro and in vivo characteristics were evaluated. Spherical RES-CMCSNPs were prepared under optimal conditions, in which average particle size, potential, drug loading and encapsulation efficiency were (155.3 ± 15.2) nm, (10.28 ± 6.4) mV, (5.1 ± 0.8)% and (44.5 ± 2.2)%, respectively. FTIR, DSC and XRD showed that RES molecules were wrapped in the nanoparticles. In vitro DPPH radical scavenging abilities showed RES-CMCSNPs were better than RES raw powder. The nanoparticles improved the solubility of RES, thereby greatly improving the antioxidant activity of the drug. In vitro release experiments of RES and RES-CMCSNPs by simulating the human gastrointestinal tract were performed, in which RES-CMCSNPs rendered better releasing effects than raw RES. Raw RES and RES-CMCSNPs results were in line with those obtained for the single-chamber model for pharmacokinetic studies in rats. Compared with the bulk drugs, the RES-CMCSNPs exhibited increased in vivo absorption, prolonged duration of action and increased relative bioavailability by 3.516 times more than those of the raw RES. In addition, the residual chloroform is less than the ICH limit for class 2 solvents.
Antioxidant, bioavailability, carboxymethyl chitosan, nanoparticles, resveratrol
Introduction Resveratrol (RES) is a natural substance and an antioxidant produced by plants to protect against environmental damage. RES is mainly derived from Polygonum cuspidatum, grapes and peanuts. In-depth studies have confirmed that this substance has a strong biological activity that yields antitumor (Wu et al., 2004), antioxidative (Meyer et al., 1997), anti-bacterial and anti-inflammatory effects; RES also provides protection against cardiovascular and hepatic diseases, and participates in immune regulation (Pace-Asciak et al., 1995). RES is a non-flavonoid polyphenol with the chemical name 3,4,5-hydroxy-1,2-stilbene and molecular formula C14H12O3 (Figure 1). RES has many beneficial physiological activities in the human body; however, this substance has low water solubility, is susceptible to oxidative decomposition and has rapid metabolism and clearance rates in vivo, which substantially influences its absorption in the body and its bioavailability, as well as limits its beneficial effects (Baur & Sinclair, 2006). Pharmacokinetic studies have shown that the Address for correspondence: Xiuhua Zhao, Associate Professor, Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, Heilongjiang, China. Tel: +86 451 82191517. Fax: +86 451 82102082. E-mail: [email protected]
History Received 26 March 2014 Revised 10 May 2014 Accepted 11 May 2014
oral bioavailability of RES is almost zero (Wenzel & Somoza, 2005). Therefore, increasing the water solubility of RES, enhancing the impact of its oral absorption and improving its bioavailability are problems that need to be addressed. Researchers have been increasingly concerned about new solubilization technologies, such as synthetic water-soluble prodrug, cyclodextrin inclusion, nanotechnology, microemulsions, liposomes and mixed micelles to improve the efficacy of poorly soluble drugs. Nanoparticle drug systems use nanoparticles as drug carriers made from natural or synthetic polymer materials. In these systems, the drug molecules covalently couple to nanoparticles by chemical methods or are wrapped and adsorbed by physical methods. Given the small size of nanoparticles, crossing biological membranes is easy, thereby effectively improving the oral bioavailability of poorly soluble drugs. This system has become important in pharmaceutical research with the development of nanotechnology. Studies have shown that the nanoparticle drug system has significant advantages, including increasing the solubility of drugs in the body, thereby enhancing absorption and improving in vivo bioavailability. This system also prolongs the effect of the drug, which achieves the slow-release effect. Drugs are controlled into specific target organs or cells upon using this system, which reduces drug side effects
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used to detect particle size, drug loading, encapsulation efficiency, physical, chemical and release characteristics through antioxidative and bioavailability studies. These technologies were also used to obtain excellent stability, high bioavailability and more desirable effects of nanoformulated oral RES and the corresponding preparation processes and quality control standards.
Materials and methods
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Figure 1. The molecular formulation of resveratrol.
(Kreuter, 2001). This system improves drug stability and prevents the drug from being degraded before reaching the lesion (Takeuchi et al., 2001). Drug absorption is also enhanced, namely, nanoparticles allow the drug to penetrate easily through biological membranes and pass through the cell gap and the blood–brain barrier to reach the diseased tissue. This system also uses carrier materials that are biodegradable and non-toxic (or less toxic). The nanoparticle drug system can be divided according to preparation process and materials, namely, nanoparticle suspensions (Elzoghby et al., 2012), solid lipid nanoparticles (Wissing et al., 2004), nanoemulsion (Tahara et al., 2011), nano-liposomes (Xiong et al., 2011) and nano-micelles (Wang et al., 2009). Biological macromolecular materials as nano-drug carriers have received increasing attention because of their renewable resources. These materials are biological adhesives having no toxicity and good biocompatibility, thereby promoting ease of degradation in vivo. Synthetic polymers and natural polymer materials are used to prepare nano-drug carriers. Examples of synthetic polymers include polyester-based aliphatic polyesters, aromatic polyesters, polycarbonates, polyanhydrides and polyamides (Panagi et al., 2001; Watnasirichaikul et al., 2002). Synthetic macromolecular materials can synthesize the desired product according to the specific need and can be modified to control the characteristics, yielding a product that is highly purified, non-toxic and readily degradable in vivo. Natural polymer materials are also commonly used as nano-drug carriers, which are classified as proteins and polysaccharides. Proteins include gelatin, serum and plant proteins; polysaccharides include cellulose, starch, sodium alginate, chitosan and cyclodextrin. Natural flora and fauna are the main sources of these materials, indicating abundant renewable resources. These materials have good biocompatibility, and can be degraded enzymatically in vivo. Natural polymer materials are ruled out in vitro from non-toxic small molecules (Horan et al., 2005) that also possess molecular chains with hydroxyl, amino and carboxyl groups used as chemical modification sites, thereby exhibiting broad applications as drug carrier materials. Carboxymethyl chitosan (CMCS), which is produced from the reaction of chitosan with chloroacetic acid in an alkaline condition, is a biological macromolecule material widely applied as a pharmaceutical carrier material (Fu et al., 2011). This substance is a bioadhesive that has good water solubility, biocompatibility, biodegradability and can promote permeability (Dong et al., 2010). In this study, CMCS was used as the drug carrier to carry RES by emulsion cross-linking. Single-factor experiments were used to optimize the preparation of nanoparticles. Physical, chemical and in vitro detection technologies were
Materials Resveratrol (purity ¼ 98%) was obtained from Shanxi Sen Fo Gao Ke Co., Ltd. (Xi’an, Shanxi, PR China). CMCS (molecular weight ¼ 14.2 104 Da) was sourced from Zhejiang Ao Xing Biotechnology Co., Ltd. (Yuhuan, Zhejiang, PR China). Tween-80, anhydrous calcium chloride, mannitol, glucose, lactose, alpha-cyclodextrin, beta-cyclodextrin and gammacyclodextrin were all obtained from Jiangsu Fengyuan Biotechnology Co., Ltd. (Suqian, Jiangsu, PR China). DPPH was from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Male rats were provided by Harbin Medical University (Harbin, Heilongjiang, PR China). Methanol, ethanol, chloroform and other reagents were of analytical grade. Preparation and optimization of RES-CMCSNPs by single-factor optimization Based on related literature and pre-experimental results, the key factors affecting the preparation of nanoparticles were CMCS concentration (in mg/ml), water:chloroform (V/V), Tween-80 ratio, homogenization time, number of highpressure homogenizers, high-pressure homogenization pressure and CaCl2 concentration. Emulsion cross-linking method was carried out stepwise using a single-factor optimization in each step to obtain the optimal conditions in preparing RESCMCSNPs. Carboxymethyl chitosan was completely dissolved in ultrapure water, and the impurities were removed by filtration. Appropriate amounts of Tween-80 were added after mixing into the CMCS solution, and a certain amount of chloroform was added dropwise to the solution by homogenization at a speed of 10 000 rpm. CMCS formed colostrum. Factors affecting emulsification of CMCSNPs included CMCS concentration (in mg/ml), water to chloroform ratio, Tween-80 ratio and homogenization time. Nanoparticle size was the indicator of the inspection. Other factors were held constant upon examination of one of the factors. CMCS concentrations varied from 2 to 6 mg/ml, with 1 mg/ml increment; water to chloroform ratios had values of 5:1, 10:1, 15:1, 20:1 and 25:1. Tween-80 ratios varied from 1/1000 to 5/1000, with an increment of 1/1000. Homogenization times varied from 1 to 9 min, with 2-min increments. Homogeneity implies that materials were refined and uniformly mixed in the homogenizing valve and in the emulsified solution after the high-pressure homogenizer, thereby obtaining more uniform, more stable and smaller nanoparticles. The main factors affecting high-pressure homogenization were homogenization pressure. Nanoparticle size was the indicator of the inspection. Homogenization pressure varied from 400 to 1200 bars, with increments of 200 bars.
Resveratrol-loaded carboxymethyl chitosan nanoparticles
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DOI: 10.3109/10717544.2014.924167
The number of heterogeneous particles varied from 3 to 15 times, with increments of 3 times. After completion of the emulsified portion, we used anhydrous calcium chloride as the cross-linking agent for ionic cross-linking. CMCS is an amphoteric electrolyte that has a negative charge under alkaline conditions. The weak acid anionic group (COO) and Ca2+ ions in inter- or intramolecular cross-linking reaction could occur to obtain nanoparticles. Added CaCl2 quality can directly affect the particle size of the nanoparticles, potential, entrapment efficiency and drug-loading amount, thereby classifying CaCl2 concentration as a factor in obtaining optimal CaCl2 qualities through single-factor experiment. Tween-80 was added to the CMCS solution and RES was sufficiently dissolved in chloroform. Chloroform was added dropwise to the CMCS solution under stirring of a homogenizer. Good emulsion was obtained after the homogenate and the highpressure homogenizer were used. CaCl2 concentrations from 1 to 6 mg/ml were selected, with a 1 mg/ml increment. CaCl2 solution was added dropwise to the emulsion until completion of the dropping. Following the completion of cross-linking, we detected the particle size and potential of each sample. Chloroform was removed using a rotary evaporator in vacuum, and was centrifuged at 10 000 rpm for 30 min. The nanoparticles were separated, the precipitate and the supernatant were collected and the precipitate was washed twice with ultrapure water. The supernatant was used to determine encapsulation efficiency and drug loading. RES-CMCSNPs were freeze-dried at 50 C for 48 h. In this process, 50% (mannitol percentage of RES-CMCS quality) mannitol was selected as lyophilized protective agent of these particles.
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Surface morphology of nanoparticles The morphology of raw RES powder, raw CMCS powder, freeze-dried RES-CMCSNPs powder and added lyoprotectant freeze–dried RES-CMCSNPs powder was ascertained through scanning electron microscopy (SEM, S4800, Hitachi, Ltd., Tokyo, Japan). After the high-pressure homogenizer was used, morphological changes of the nanoparticles were analyzed via electron microscopy (BH-2, Olympus Corporation, Tokyo, Japan). Chemistry characterization of nanoparticles The molecular structures of RES raw powder and nanoparticles were examined with Fourier transform infrared (FTIR) spectroscopy using a MAGNA-IR560 E.S.P (Nicolet Pittsburgh, PA, USA) instrument. The spectra were obtained in transmission mode at room temperature in 4000–400 cm1 range at a resolution of 2 cm1. Physical status of RES in RES-CMCSNPs An X-ray diffractometer (Philips, Xpert-Pro, The Netherlands) was used to determine the physical status of paclitaxel in the nanoparticles. The diffraction angle (2y) was recorded from 3 to 80 with a scanning speed of 5 /min. The samples were irradiated using a Cu target tube at 30 mA current and 40 kV voltage. Thermal analysis was performed with differential scanning calorimetry (DSC, TA Instruments, model DSC 204, Woodland, CA, USA). All thermal analyses were performed for 5.0 mg samples at a heating rate of 10 C/min and a temperature range of 20–300 C.
Characterization of RES-CMCSNPs
GC analysis
Size, zeta potential, drug encapsulation and loading efficiency
The residual chloroform in the RES-CMCSNPs were analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with DB-WAX polyethylene glycol capillary column (30.0 m 250 mm 0.25 mm, nominal) equipped with a G1540N-210 FID detector (Agilent Technologies, San Jose, CA, USA). Peaks areas were used for obtaining quantitative data. The conditions of GC analysis of chromatograph were as follows: oven temperature was maintained at 40 C for 5 min initially, and then raised at the rate of 10 C/min to 200 C, which was maintained for 3 min at last. The injector and the detector temperatures were set 200 C and 250 C, respectively. Nitrogen was used as carrier gas at a flow rate of 25 ml/ min, and 5 ml samples were injected manually in the split mode with a split ratio 25:1. Hydrogen gas and air flow rate were 30 and 400 ml/min.
The mean particle size and electric potential of the obtained nanoparticles were analyzed by dynamic light scattering equipment (ZetaPALS, Brookhaven Instruments, Long Island, NY, USA). The samples were prepared by dissolving the powdered nanoparticles with deionized water, and the suspension was sonicated for a while. Each experimental preparation was executed in triplicate, and data were obtained from the average of several measurements. Free RES in the supernatant was tested by high-performance liquid chromatography (HPLC) using a Diamonsil C18 column (250 mm 4.6 mm, 5 mm; Dikma Technologies, Beijing, China). An HPLC system (Waters Corporation, Milford, MA, USA), consisting of a Waters 600 Controller equipped with a Waters 717 plus autosampler, and a Waters 2487 UV detector were used. Ten microliters of the sample was injected into a C18 column at 25 C, with a 40:60 water (0.25% acetic acid)/ methanol mobile phase. The elution flow rate was 1 ml/min, and detection was accomplished at 306 nm. DEE and DLE were calculated using the following previously reported equations: DEE¼
ResðtotalÞ ResðfreeÞ 100% ResðtotalÞ
ð1Þ
DLE¼
ResðtotalÞ ResðfreeÞ 100% RES CMCSNPs
ð2Þ
DPPH radical scavenging activity assay of RES-CMCSNPs In this assay, 25.76 mg DPPH powder was added to 50% ethanol solution to yield a concentration of 6.5 104 mol/l of DPPH stock solution. Three samples were employed: the RES raw powder group, the RES-CMCSNPs group containing the same quality of RES and the CMCSNPs group with the same quality of that in RES-CMCSNPs. Each group used PBS (4 mM, pH ¼ 7.4) to configure different concentration
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gradients into five samples (0.0625, 0.125, 0.25, 0.5 and 1 mg/ ml). Up to 3.5 ml of DPPH radical solution was mixed with 0.5 ml of three sample groups at various concentrations. Absorbance at 517 nm was measured after 60 min in an incubator at 37 C, in which each absorbance value was measured in triplicate. PBS was employed as blank control. DPPH radical scavenging ability was calculated as Scavenging ability% ¼
A0 As 100 As
ð3Þ
where A0 is the absorbance of the control and AS is the absorbance of the tested sample.
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In vitro RES release from RES-CMCSNPs The condition of in vitro oral drug release was at 37 C, thereby using artificial gastric juice and intestinal fluid to simulate the human body environment. One thousand milliliters of artificial gastric juice and intestinal juice were used according to the Pharmacopoeia records. RES was not conducive for detection because of insolubility in water. We added 1 ml of Tween-80 into the artificial gastric and intestinal fluids to allow more uniform dispersion dissolution of RES. Five milligrams of RES raw powder and lyophilized RESCMCSNPs powder containing the same quality of RES were dissolved in 10 ml of artificial gastric and intestinal fluids, and were placed in dialysis bags that were, respectively, suspended in two 250-ml beakers containing 200 ml fluids. The beakers were placed on magnetic stirrers and reacted at a constant temperature of 37 C. The speed was maintained at 50 rpm according to the in vivo rate of gastric peristalsis. At predetermined time intervals, 3 ml dialysate was withdrawn for detection, and equal volumes of fresh artificial gastric and intestinal fluids were added each time. The concentration of RES was determined by highperformance liquid chromatography. Liquid detection conditions corresponded with previously described conditions. Cumulative release percentage was calculated with the following formula: Ci0 ¼ C1 0 ¼ Ciþ1 Ciþ1 i P
Qi ¼
i¼1
ð4Þ
ðV Vi Þ Ci V
ð5Þ
Ci0 V M
100%
ð6Þ
where Ci is the RES concentration of each sample withdrawn at predetermined time intervals, Ci0 is the increase in RES concentration during each time interval, V is the volume of the release buffer, Vi is the volume of each withdrawn sample, M represents the RES loaded in the RES-CMCSNPs and Qi represents the accumulative release percentage at a predetermined time point. Drug release studies were conducted in triplicate for each sample. In vivo bioavailability studies of RES-CMCSNPs in rats Six healthy male SD rats were used and divided into two groups according to similarity in weight. Three parallel tests
were performed for three rats of each group. Rats were fasted for 12 h before administration. RES raw powder (control group) and RES-CMCSNPs (experimental group) were administered orally to the rats, with 50 mg/kg dosage (calculated by RES). After administration, 1 ml of blood was collected from the fundus after administration. Sampling times for the control group were 0.5, l, 2, 3, 4, 5, 6, 8, 12 and 16 h, and that for the experimental group were 0.5, l, 2, 3, 4, 5, 6, 8, 12, 24 and 48 h. Blood was placed in a heparinized centrifuge tube, and was centrifuged for 5 min at 3500 rpm. Upper plasma was taken following the centrifugation. One hundred microliters of plasma was placed in a 1.5-ml Eppendorf tube, followed by the addition of 300 ml methanol. After vortex mixing for 3 min and centrifugation for 10 min at 10 000 rpm, the upper organic layer was drawn to dry with nitrogen in a water bath at 40 C. The residue was dissolved with 100 ml methanol, and 20 ml of the supernatant was taken for injection. In this process, the methanol:water (0.25% acetic acid) mobile phase was 60:40 (v/v), the flow rate was 1 ml/min, the column temperature was 30 C, the detection wavelength was 306 nm and the injection volume was 20 ml.
Results and discussion Preparation and optimization of RES-CMCSNPs Emulsification of CMCSNPs Figure 2(a) shows that the concentration of CMCS had a marked effect on the particle size of CMCSNPs upon fixing three other factors. The particle size of CMCSNPs increased with the concentration of CMCS, which was attributed to the increase in viscosity upon the increase in CMCS concentration, which easily agglomerated the particles and resulted in increased particle size. If the particle size of CMCSNPs was too small, the nanoparticles were more difficult to separate. Moreover, absorption would be affected if the particle size was too large. The drug could not be fully contacted if the concentration of CMCS was relatively low, thereby affecting the drug loading. Thus, we chose CMCS concentration of 5 mg/ml as the optimization condition. Figure 2(b) shows that the volume ratio of water and chloroform likewise significantly affected the particle size of CMCSNPs. The particle size of CMCSNPs increased with volume ratio of water and chloroform, whereas three other factors were held constant. Based on particle size, the 10:1 volume ratio of water and chloroform was determined as the optimal condition. Figure 2(c) shows that as the three other factors were held constant, the ratio of Tween-80 and water had no significant effects on the particle size of CMCSNPs, which was 150 nm. As additives to drugs, the proportion of Tween80 would affect the human body. Taking safety into account, we chose 1/1000 as the optimization criteria. Figure 2(d) shows that the homogenization time of CMCSNPs also affected particle size, with the three other factors held constant. From 1 to 3 min, the particle size decreased with the increase of homogenization time because a short time led to an uneven emulsification. Some particles agglomerated together, resulting in a slightly larger particle size. The particle size decreased with the increase of the
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DOI: 10.3109/10717544.2014.924167
Resveratrol-loaded carboxymethyl chitosan nanoparticles
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Figure 2. Impact trend graph of: (a) CMCS concentration, (b) the volume ratio of water and chloroform, (c) the ratio of Tween-80 and water, (d) homogenized time, (e) the pressure of high-pressure homogenization and (f) the time of high-pressure homogenization to CMCSNPs size.
homogenization time. In the following 3–9 min, the particle size of CMCSNPs increased with the homogenate time, because the emulsification time was longer and mechanical stirring produced mechanical energy that increased the energy of the. To reduce this energy, the particles should be closer to each other, thereby reducing the specific surface area and increasing the particle size. Based on changes in particle size, we selected 5 min as the appropriate time of emulsification. Figure 2(e) shows that pressure affects the size of CMCSNPs during high-pressure homogenization. When the number of homogenization was fixed, increasing the pressure initially reduced the size of the nanoparticles because the increased homogenization pressure produced a large mechanical force. The particle size was too large or uneven, and gradually became smaller and more uniform after homogenization. When the pressure exceeded 600 bar, the particle size increased with pressure.
Figure 2(f) shows that when the homogenization pressure was fixed, the particle size decreased with increasing number of homogeneous particles because this increase lengthened the mechanical action time. The particles reduced after homogenization. When the number of homogeneous particles constantly increased, the particle size also increased High-pressure homogenization resulted in reduced and uniform particle size. However, the size of nanoparticles increased when the pressure and number of homogenization continued to increase up to a certain value because the pressure was high or homogenization was performed too many times. Aggregation between the particles repeatedly increased the average particle size. The limited emulsifier could not be effectively adsorbed to the particle surfaces, thereby reducing the emulsification, aggregating the droplets, increasing the particle size and causing instability. Ultimately, we chose 600 bar as the pressure of high-pressure
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Figure 3. Impact trend graph of CaCl2 concentration to size, zeta potential, drug loading and encapsulation efficiency of RES-CMCSNPs. (a) Size, (b) zeta potential, (c) drug loading and (d) encapsulation efficiency.
homogenization, and six times as the optimal number of homogenization times. Ionic cross-linking Figure 3 shows that at constant volume, CaCl2 concentration had varying effects on size, zeta potential, drug loading and encapsulation efficiency of RES-CMCSNPs. Figure 3(a) reveals that particle size increased with CaCl2 concentration because the increase of Ca2+ had ionic cross-linking with negatively charged groups (COO) on CMCS, thereby allowing more CMCS molecules to combine and yield a larger particle size. The emulsions were negatively charged prior to the addition of CaCl2. When the increased Ca2+ cross-linked together with negative charges, the negative charges of the emulsion decreased. Figure 3(b) shows that the zeta potential of RES-CMCSNPs increased with CaCl2 concentration. Figure 3(c) and (d) shows that drug loading and encapsulation efficiency increased with CaCl2 concentration. When this concentration reached 5 mg/ml, the drug loading and encapsulation efficiency were, respectively, 5.1% and 44.3% of the maximum. Further increasing this concentration to 6 mg/ml stopped the increase of both drug loading and encapsulation efficiency. When the CaCl2 concentration was 5 mg/ml, Ca2+ and CMCS quality reached a suitable ratio, which achieved an optimal state for the Ca2+ and CMCS cross-linking. At this point, the particle size and potential were the most stable and appropriate, in which the drug
loading and encapsulation efficiency reached maximum values. Therefore, we selected the CaCl2 concentration of 5 mg/ml as the optimum condition for ionic cross-linking. Characterization of RES-CMCSNPs Size, zeta potential, drug encapsulation and loading efficiency According to the optimal formulation, validation tests were carried out in triplicate. Results showed that the average nanoparticle size was (155.3 ± 15.2) nm, the potential was (10.28 ± 6.4) mV, drug loading was (5.1 ± 0.8)% and encapsulation efficiency was (44.5 ± 2.2)%. The batch reproducibility was good. Surface morphology of nanoparticles An optical microscope was used to observe and compare the morphologies of RES-CMCSNPs. Figure 4(a) shows the morphology of RES-CMCSNPs without homogenization in different multiples. The particle sizes were relatively large and uneven, and a two-layer structure could be observed. The outer layer should be aqueous phase with CMCS, and the inner is the oil phase containing RES, thereby successfully proving the initial emulsion. Figure 4(b) shows the morphology of RES-CMCSNPs after homogenization in different multiples. Compared with that shown in Figure 4(a), the particle size was significantly smaller and became more uniform after high-pressure homogenization.
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DOI: 10.3109/10717544.2014.924167
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Figure 4. Different multiples morphology of RES-CMCSNPs: (a) without homogenization (10 10) and (b) after homogenization (10 10).
Figure 5. Scanning electron micrograph: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 5 shows the scanning electron micrograph of RES raw powder, CMCS, RES-CMCSNPs and RES-CMCSNPs with mannitol. Figure 5(a) and (b) shows that the RES raw powder exhibited a needle crystal structure, and raw CMCS powder was in amorphous powder state. Figure 5(c) shows
that RES-CMCSNPs were spherical and have relatively uniform sizes. Figure 5(d) shows that mannitol as the freezing-drying protective agent has a significant function in protecting RES-CMCSNPs as the nanoparticles were embedded.
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Figure 6. Infrared spectrogram: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 7. XRD pattern: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs, (d) RES-CMCSNPs with mannitol and (e) mannitol.
Chemistry characterization of nanoparticles
XRD pattern of RES-CMCSNPs with mannitol, in which peaks appeared at 6.5 , 16.2 and 19.0 , indicating the crystal structure of mannitol. Figure 7(e) shows the XRD pattern of mannitol, in which peaks appeared at 14.8 , 19.0 and 23.5 , which indicate the crystal structure of mannitol. Figure 8(a) represents the DSC thermogram of RES raw powder. An obvious endothermic peak existed when the temperature increased to 267.82 C, which was very close to the melting point of RES, indicating that RES crystals have melted. Figure 8(b) shows that CMCS has no obvious endothermic or exothermic peak, proving that CMCS has amorphous state structures. Figure 8(c) shows the DSC thermogram of RES-CMCSNPs, which is very similar to that of CMCS. No apparent melting processes were observed, which revealed that RES-CMCSNPs have an amorphous state structure. Figure 8(d) shows the DSC thermogram of RESCMCSNPs with mannitol. A crystal fusion endothermic peak exists when the temperature has reached 164.4 C, indicating the crystal structure of mannitol.
Figure 6(a) shows the infrared spectrogram of the RES raw powder. A phenolic hydroxyl group absorption peak at 3249 cm1 and benzene ring absorption peaks at 1622, 1540 and 1506 cm1 exist. Figure 6(b) shows the infrared spectrogram of CMCS, in which a hydroxyl group absorption peak at 3424 cm1 exists. Figure 6(c) shows the infrared spectrum of RES-CMCSNPs. The characteristic absorption peak appeared at the same position, but the characteristic absorption peak of RES did not appear upon comparison with Figure 7(b), proving that RES had been completely wrapped in CMCS. Figure 6(d) shows the infrared spectra of RES-CMCSNPs with mannitol. In addition to the characteristic absorption peaks of CMCS, the characteristic peaks of mannitol were also markedly observed. Physical status of RES in RES-CMCSNPs Figure 7(a) shows the XRD pattern of RES raw powder. Multiple characteristic peaks for RES existed because of the crystalline structure. Figure 8(b) shows the XRD pattern of CMCS, in which characteristic peaks from 5 to 50 were not observed, indicating that CMCS has amorphous structures. Figure 7(c) shows the XRD pattern of RES-CMCSNPs, which is similar to that in Figure 7(b), implying that RES is almost completely encapsulated in CMCS. Figure 7(d) shows the
Solvent residue analysis The problem of solvent residues is also under consideration in pharmaceutical products. In this article, emulsion crosslinking method was carried out to paper RES-CMCSNPs. Figure 9(a) and (b) shows the results of chloroform residue
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DOI: 10.3109/10717544.2014.924167
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Figure 10. DPPH radical scavenging rate. (a) RES raw powder, (b) RES-CMCSNPs with CMCSNPs as control, (c) RES-CMCSNPs and (d) CMCSNPs.
DPPH radical scavenging activity assay of RES-CMCSNPs
Figure 8. DSC thermogram: (a) RES raw powder, (b) CMCS, (c) RES-CMCSNPs and (d) RES-CMCSNPs with mannitol.
Figure 10(a), (c) and (d) shows the DPPH radical scavenging ability of RES raw powder, RES-CMCSNPs and CMCSNPs. Figure 10(b) is the practically measured result of the DPPH radical scavenging ability of RES-CMCSNPs with CMCSNPs as control. As seen from Figure 10(c), CMCSNPS itself have little antioxidant activity. In the range of 62.5–1000 mg/ml, the clearance rate also gradually increased with the concentration of RES. When the concentration was 1000 mg/ml, the clearance rate reached almost 70%. The DPPH radical scavenging capacities of RES-CMCSNPs were considerably better than those of the RES raw powder. When the samples reacted with the DPPH radical, 50% ethanol solution was introduced to improve the solubility of RES. Thus, minimal difference existed between low-concentration samples of RES raw powder and RES-CMCSNPs. When the concentration was 1000 mg/ml, the DPPH radical scavenging rate of RESCMCSNPs was slightly higher than that of RES raw powder. DPPH radical scavenging experiment showed that the antioxidant activity of RES substantially improved, which possible reason is that RES-CMCSNPs improved the watersolubility of RES raw powder. In vitro RES release from RES-CMCSNPs
Figure 9. Gas chromatograms of samples: (a) chloroform standard solution and (b) RES-CMCSNPs.
using the GC method. From the chromatograms of chloroform standard solution, a regression equation between peak area (Y) and ethanol concentration (x) can be fitted as Y ¼ 127.1015 x + 0.6195 (R2 ¼ 0.9998). The linear range of chloroform was 0.0015625–0.1 mg/ml. According to the regression equation, the residual chloroform content in RES-CMCSNPs is 60 ppm. Since the ICH limit for chloroform in class 2 solvents is 60 ppm or 0.006%, the RES-CMCSNPs met ICH requirements and are suitable for pharmaceutical use.
Figure 11(a) and (b), respectively, represents the release profiles of RES raw powder and RES-CMCSNPs in a simulated gastric fluid. Figure 11(a) shows that RES raw powder is quickly released within the first 2 h when the cumulative release percentage has reached 26.9% because of a small amount of the raw powder that is completely dissolved and has a quick release process. At a later time, RES raw powder is slowly released until 68 h when the cumulative release percentage has reached only 65.9%. However, the RES raw powder was inadequately dissolved in the dialysis bag because of the poor solubility of RES raw powder in water. Upon the release of RES from the dialysis bag, the degree of saturation changed. Undissolved RES gradually dissolved in the dialysis bag and was continuously released. Thus, the
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Table 1. Pharmacokinetic parameters after gavage. Atrioventricular parameters
RES
RES-CMCSNPs
A (mg/ml) Ke (1/h) Ka (1/h) t1/2 (ka) (h) t1/2 (ke) (h) Tmax (h) Cmax (mg/ml) AUC ((mg/ml)h) CL/f(s) (mg/h/(mg/ml)) V/f(c) ((mg)/(mg/ml))
3.95 0.13 0.62 1.12 5.44 3.21 2.00 24.60 0.41 3.19
3.19 0.03 0.39 1.76 20.55 6.83 2.32 86.50 0.12 3.43
Figure 11. Release profile in simulated gastric fluid. (a) RES raw powder and (b) RES-CMCSNPs.
Figure 13. Concentration–time curve. (a) RES raw powder and (b) RES-CMCSNPs.
Figure 12. Release profile in simulated intestinal fluid. (a) RES raw powder and (b) RES-CMCSNPs.
cumulative release percentage was initially low but gradually increased. Figure 11(b) shows the RES-CMCSNPs release profile divided into two phases: burst release and sustained release periods. The first 1 h was burst release period because of a small amount of drug loosely bound in the surface of nanoparticles, causing rapid drug release. Thereafter, during the sustained period, RES wrapping in nanoparticles became more difficult through microporous diffusion, and the release depends on the degradation of CMCS. At 44 h, the cumulative release percentage reached a maximum of 82.8%, indicating that the RES-CMCSNPs were almost completely released. The cumulative release percentage reached 81.3% in 68 h, and was higher than that of RES raw powder. Figure 12(a) and (b), respectively, represents the release profiles of RES raw powder and RES-CMCSNPs in simulated intestinal fluid, which were similar to the release in simulated gastric fluid. RES raw powder had a burst release process in the first 2 h, and the cumulative release percentage reached 33.7%. When released at 68 h, the cumulative release percentage reached 64.8%. Slow release was observed, and the dialysis bag still contained the undissolved RES raw
powder. By contrast, the cumulative release percentage of RES-CMCSNPs reached 34.1% within 1 h. When time reached 44 h, the cumulative release percentage reached a maximum of 80.5%, indicating that the RES-CMCSNPs were almost completely released. The cumulative release percentage reached 79.2% in 68 h, and was higher than that of the RES raw powder. The drug release profile was analyzed with OriginPro 8.5.1 software (Northampton, MA, USA). In the simulated gastric juice, release characteristics of the RES raw powder agreed withx the Higuchi equation y ¼ 30:93 x e¼17:68 36:84e1:57 þ 65:12 (R2 ¼ 0.990). The same trend was observed xfor the release curve of RES-CMCSNPs: x y ¼ 48:61e7:95 36:11e0:44 þ 80:90 (R2 ¼ 0.993). The release curves for RES raw powder and RES-CMCSNPs in the intestinal fluid also agree withxthe Higuchi equation, given x by y ¼ 27:85ex20:66 40:90ex1:01 þ 64:66 (R2 ¼ 0.995) and y ¼ 51:09e8:47 31:044e0:5 þ 80:66 (R2 ¼ 0.996). In vivo bioavailability studies of RES-CMCSNPs in rats The 3p87 software (Chinese Pharmacological Society, Beijing, PR China) was used to model fit the data, in which we obtained various kinetic parameters of RES raw powder and RES-CMCSNPs (Table 1). They were single-room models. The weighing coefficients were both 1/C/C. Figure 13 shows the plasma concentration–time curve.
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DOI: 10.3109/10717544.2014.924167
After oral administration, in vivo absorption and elimination of RES raw powder were rapid, in which t1/2 (ka) was 1.12 h, t1/2 (ke) was 5.44 h, peak time was 3.21 h, maximum plasma concentration was 2.00 mg/ml and the area under the concentration–time curve AUC was 24.60 (mg/ml)h. After oral administration of RES-CMCSNPs, half-life t1/2 (ka) extended to 1.76 h, elimination half-life t1/2 (ke) extended to 20.55 h, peak time reached 6.83 h, maximum plasma concentration increased to 2.32 mg/ml and the area under the concentration–time curve for AUC increased to 86.50 (mg/ ml)h, was 3.516 times that of RES. The relative bioavailability of RES-CMCSNPs was 3.516 times as large as RES. The maximum plasma concentration time of nanoparticles was later than that of the RES raw powder, which further proved that nanoparticles exhibited sustained release effect, thereby extending the drug peak time. The drug was wrapped in CMCS, which prevented the removal of the drug from the body, and induced the clearance rate to decrease and the elimination half-life to significantly increase. These phenomena extended the presence time of the drug in the body and contributed to the improvement of bioavailability. Comparing the concentration–time curve and the kinetic parameters, the bioavailability of nanoparticles in rats substantially improved. One reason may be the improvement of RES water solubility. Another reason may be that particle size was suitable for uptake by intestinal cells and CMCS as a drug carrier, endowing the nanoparticles with biological adhesion and promoting permeability advantages, thus extending the gastrointestinal residence time of RES and aiding in the absorption of drugs.
Conclusions This study attempts to improve the oral bioavailability of RES, and CMCS as a drug carrier. RES nanoparticles were prepared by emulsion cross-linking. Single-factor experiment was used to obtain the optimal conditions for nanoparticles. RES-CMCSNPs were spherical with uniform particle size distribution, and RES was completely wrapped in the nanoparticles. Oxidation in vitro experimental tests showed that the nanoparticles improved the water solubility of RES, thereby improving the antioxidant activity of the drug. In vitro release test showed the good release effect of RESCMCSNPs. In vivo bioavailability study of drugs showed that nanoparticles had better absorption in the body, in which the relative bioavailability was increased 3.516 times compared with the RES raw powder. Results indicated that the nanoparticle drug system promoted the absorption of RES, improved the raw powder which was initially insoluble in water and improved its oral bioavailability. In addition, the residual chloroform is less than the ICH limit for class 2 solvents. In summary, this article provides a theoretical and experimental basis for solving poor water solubility and low oral bioavailability of RES.
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Acknowledgements The authors are grateful for the precious comments and careful corrections made by anonymous reviewers.
Declaration of interest The authors would also like to acknowledge the financial support from the Fundamental Research Funds for the Central Universities (DL12EA01-02) and the National Natural Science Foundation of China (No. 21203018).
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