PEGylated Solanesol for Oral Delivery of Coenzyme Q10

Apr 18, 2017 - ABSTRACT: Coenzyme Q10 (CoQ10) is widely used in preventive or curative treatment of cardiovascular diseases. However,. CoQ10 exhibits ...
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PEGylated Solanesol for Oral Delivery of Coenzyme Q10 Benkai Qin, Lei Liu, Yangyang Pan, Yingchun Zhu, Xiaohe Wu, Shiyong Song,* and Guang Han* Institute of Pharmacy, Pharmacy College of Henan University, Jinming, Kaifeng, Henan 475001, China S Supporting Information *

ABSTRACT: Coenzyme Q10 (CoQ10) is widely used in preventive or curative treatment of cardiovascular diseases. However, CoQ10 exhibits an extremely low solubility in aqueous medium as well as a poor oral bioavailability. Therefore, solanesyl poly(ethylene glycol) succinate (SPGS) and CoQ10 were formulated as CoQ10-SPGS micelles with a high content of CoQ10 to improve the bioavailability of CoQ10 in rat. Findings indicate that, in the CoQ10-SPGS micelles, SPGS is self-assembled into stable nanosized micelles with a CoQ10 loading capacity of more than 39%. The CoQ10-SPGS micelles exhibit an enhanced photostability upon exposure to simulated sunlight. In vivo experiments demonstrate that, as compared to that of the coarse suspensions of CoQ10, there was three-fold enhancement of oral bioavailability for CoQ10-loaded SPGS micelles depending on varying molecular weight of SPGS. In the encapsulation of CoQ10 by SPGS micelles, the self-assembled nanocarriers with strong muco-adhesive properties lead to increases in the solubility and oral absorption of lipophilic CoQ10 nanoparticles. KEYWORDS: coenzyme Q10, solanesol, micelles, water dispersibility, photostability, bioavailability

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Liu et al.20 employed Kollipher HS15 as a carrier to build up a stable micelle for the delivery of CoQ10. They reported that the CoQ10-loaded micelles obtained under the optimized conditions exhibit an entrapment efficiency of 99.36% and drug loading content of 13.77%, showing potential as a drug candidate for further clinical applications. PEGylated α-tocopherol(vitamin E), that is, α-tocopheryl polyethylene glycol succinate (TPGS), is another safe pharmaceutical adjuvant that has been widely applied in developing various drug delivery systems.23 TPGS is able to form micelle structures to improve solubilization of hydrophobic drugs, and it is also an inhibitor of P-glycoprotein to overcome P-glycoprotein mediated multidrug resistance.24 It has been found that TPGS-based nanoparticles could significantly extend the half-life of the formulated drug in the plasma.25,26 Zhou et al.27 prepared TPGS-based CoQ10-loaded nanoemulsions by hot-high-pressure homogenization. They found that the TPGS-based nanoemulsions can more efficiently deliver CoQ10 to heart tissue than lecithin-based nanoemulsion. However, TPGS exhibits a high critical micelle concentration (CMC); hence, it generally needs to be mixed with other amphiphilic materials to increase stability and solubilization capacity.28 Alpha-tocopherol based amphiphilic materials for delivery of hydrophobic therapeutic agents retain the advantages of vitamin E, such as biocompatibility, bioactivity, and hydrophobicity (affinity to hydrophobic molecules), and they can extend the half-life of the drug in plasma and enhance the cellular uptake of the drug. Moreover, the derivatives of natural materials, such as TPGS, PEGylated phosphatidylethanolamine (PEG−PE),29 and PEGylated distearoylphosphatidylethanolamine (PEG-DSPE),30,31 have

INTRODUCTION Coenzyme Q10 (CoQ10), an endogenously produced lipophilic antioxidant, exists in the cells of heart, liver, and skeletal muscles,1 and it plays a crucial role in various essential cellular processes.2,3 A deficiency of CoQ10 can result in various types of myopathy and neuropathy, and the exogenous supplementation of CoQ10 is of significance in promoting cardiovascular health, combating aging, supporting healthy blood glucose levels, and improving neurodegenerative diseases.4−6 However, CoQ10 is lipophilic and exhibits a poor solubility in aqueous media ( 10) as well as a low bioavailability in human gastrointestinal tract.7 Therefore, it is imperative to search strategies to enhance the water dispersibility and oral bioavailability of CoQ10. In this respect, cyclodextrin complexes,8,9 nanoemulsions,10,11 solid dispersion,12 polymeric nanoparticles,1,13 polymeric micelles,14 microemulsions,15 lyotropic liquid crystalline nanoparticles,16 and self-emulsifying drug delivery systems are of significance.17 Unfortunately, they often rely on cosolvents or surfactants that cause toxicity and other undesirable side effects. In addition, the moderate stability of CoQ10 products needs to be improved to delay its photosensitive degradation. Among various approaches for enhancing the solubility and stability of CoQ10, the formulation of micelles is promising. The micelles of amphiphilic block copolymers could have promising potentials in biomedical applications,18 because they consist of a hydrophobic core and hydrophilic shell, which can encapsulate poorly water-soluble drugs and provide aqueous solubility. Also, they have minimum toxicity and other undesirable side effects because of the exclusion of cosolvents and the inclusion of biocompatible micelle-forming materials. The nonionic surfactant Kollipher HS15, a polyglycol monoester and diester of 12-hydroxystearic acid with about 30% free polyethylene glycol, is a good example of the abovementioned amphiphilic block copolymer, and its micelle is able to enhance the solubility of poorly soluble drugs19−21 and inhibit the P-glycoprotein efflux mechanism to some extent.22 © 2017 American Chemical Society

Received: Revised: Accepted: Published: 3360

January 12, 2017 April 5, 2017 April 11, 2017 April 18, 2017 DOI: 10.1021/acs.jafc.7b00165 J. Agric. Food Chem. 2017, 65, 3360−3367

Article

Journal of Agricultural and Food Chemistry

Laboratory Animal Center (Zhengzhou, China). The animal procedures were approved by the Animal Experimentation Ethics Committee of Henan University (permission number HUSAM2014− 216) and were performed strictly according to the Guide for the Care and Use of Laboratory Animals and the Regulation of Animal Protection Committee. Synthesis of Solanesyl Poly(ethylene glycol) Succinate (SPGS). Synthesis of Monosolanesol Solanesyl Succinate (MSS). The route reported elsewhere was adopted to synthesize MSS.38 Briefly, succinic anhydride (0.96 g, 9.51 mmoL) and triethylamine (1.00 mL) were dissolved in anhydrous dimethylformamide (15.00 mL). Then solanesol (4.00 g, 6.34 mmoL) and DMAP (0.39 g, 3.17 mmol) dissolved in anhydrous dichloromethane (20.00 mL) were added into the dimethylformamide solution of succinic anhydride and triethylamine. The resultant mixed solution was allowed to react at room temperature for 48 h under the protection of nitrogen gas. Upon completion of the reaction, the mixed solution was washed with distilled water and concentrated by rotary evaporation to afford the crude MSS. The crude product was further purified by a chromatography on length 25 cm, diameter 24 mm silica gel column with n-hexane/ethyl acetate (1:1, v/v) to provide a faint yellow solid, the final target product MSS (yield, 70 wt %; molecular weight, 713.14). The nuclear magnetic resonance (1H NMR) spectrum of MSS was recorded with a AV-400 MHz spectrometer (Bruker, Karlsruhe, Germany); solvent: CDCl3. 1H NMR δ: 5.34 (t, 1, J = 8.8 Hz, −CH2CC−H), 5.12 (t, 1, J = 8.4 Hz, CH3CH3CC−H), 4.61 (d, 2, J = 9.2 Hz, −COOCH2−), 2.65 (m, 4, J = 6.8 Hz, −OOCCH2CH2COO−), 1.98−2.06 (m, 4, J = 8.8 Hz, −C CCH2CH2CC−), 1.68−1.70 (d, 6, CH3CH3CC−), 1.60 (s, 3, CH3−CC−). Synthesis of SPGS. Hydrophobic MSS was covalently conjugated to the mPEG backbone by esterification reaction. Briefly, MSS (1.17 g, 1.60 mmoL), DMAP (0.234 g, 1.92 mmoL), and DCC (0.396 g, 1.92 mmoL) were dissolved in 30 mL of dichloromethane and stirred for 1 h to activate the carboxyl groups. Then the mPEG (8.0 g, 1.60 mmoL) was added to the mixed solution and allowed to react at room temperature for 48 h. Upon completion of the reaction, the reaction system was filtered to remove DCU and the filtrate concentrated by rotary evaporation. The concentrated filtrate was purified with a Sephadex LH-20 column chromatograph (methanol/trichloromethane = 2:1, v/v) to afford SPGS products with a molecular weight of 2000 and 5000 Da (denoted as SPGS-2K and SPGS-5K) in the same yield of 75.6 wt %. 1H NMR (400 MHz, CDCl3) δ: 5.34 (t, 1, J = 8.8 Hz, −CH2CC−H), 5.12 (t, 1, J = 8.4 Hz, CH3CH3CC−H), 4.61 (d, 2, J = 9.2 Hz, −COOCH2−), 4.25 (t, 2, J = 4.4 Hz, −CH2OOC−), 3.70 (s, 4, −CH2CH2−O−), 3.40 (s, 3, CH3O−), 2.65 (m, 4, J = 6.8 Hz, −OOCCH2CH2COO−), 1.98−2.06 (m, 4, J = 8.8 Hz, −C CCH2CH2CC−), 1.68−1.70 (d, 6, CH3CH3CC−), 1.60 (s, 3, CH3−CC−). Preparation of Blank SPGS Micelles and Analysis of Their Particle Size. SPGS micelles were prepared by solvent evaporation method. Briefly, SPGS (40 mg) was dissolved in acetone (1 mL). The resultant solution was added dropwise into 20 mL of pure water and stirred at room temperature for 24 h to afford SPGS micelles. The SPGS micelle formulations were filtered through 0.45 and 0.22 μm syringe filters. The particle size and distribution of the SPGS micelles were measured with a Zetasizer Nano-ZS90 particle size/zeta potential analyzer (Malvern Instruments, UK). Colloidal storage stability and dilution stability of the micelle formulations were evaluated by dynamic light scattering (DLS) measurements. Prior to DLS measurements, the prepared micelle formulations were stored at 4 °C or at ambient room temperature (25 °C) for 30 d. The drug-loaded micelles were diluted with deionized water at a volume ratio of 1:9, 1:49, 1:199, and 1:999 to afford the samples for diluted stability evaluation and particle size measurements as well. Three repeat measurements were conducted for each diluted micelle and the averaged particle size recorded. CMC Measurement. The CMC of the SPGS micelles was determined with pyrene as the fluorescence probe, where 0.5 mL of the stock solution of pyrene in acetone (6.0 × 10−6 mol/L) was

shown great potential as efficient drug delivery systems under various conditions. Solanesol (Figure 1) is well-known as a critical intermediate in the synthesis of ubiquinone drugs (e.g., CoQ10).32−34

Figure 1. Synthesis of amphiphilic diblock copolymers containing solanesol and PEG. Conditions and reagents: (1) triethylamine, anhydrous dimethylformamide, and anhydrous dichloromethane, room temperature, 48 h; (2) DCC, DMAP, room temperature, 48 h.

Extracted from tobacco plants and composed of 45 carbon atoms, solanesol possesses antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and antiulcer activities,35,36 and it also could effectively absorb ultraviolet radiation, scavenge lipid free radicals, block lipid peroxidation, and inhibit the activity of tyrosinase.37 Here, we report a new biocompatible amphiphilic material, solanesol poly(ethylene glycol) succinate (SPGS), for the oral delivery of CoQ10. It is anticipated that the integration of solanesol and CoQ10 with structural similarity would provide good chances to enhance the water dispersibility and photostability of CoQ10, thereby achieving a high drug loading capacity in the SPGS micelles.

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MATERIALS AND METHODS

Materials. Analytical grade reagents mPEG (ethyl poly(ethylene glycol); Mn = 2K and 5K), CoQ10, and 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Succinic anhydride was commercially obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Solanesol, N,N′-dicyclohexylcarbodiimide (DCC), methyl 4hydroxybenzoate, and 4-dimethylaminopyridine (DMAP) were purchased from Shanghai Darui Fine Chemical Ltd. (Shanghai, China). Vitamin K1 was provided by Aladdin (Shanghai, China). The human L02 hepatocytes (HL-7702) and human liver carcinoma cells (HepG-2) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Animals. Female Sprague−Dawley rats (SD rats) of 220 ± 30 g and 8−9 weeks old were obtained from Henan Provincial Medical 3361

DOI: 10.1021/acs.jafc.7b00165 J. Agric. Food Chem. 2017, 65, 3360−3367

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Journal of Agricultural and Food Chemistry

Figure 2. 1H NMR spectra of compound (A) MSS, (B) SPGS-2K, and (C) SPGS-5K. Photostability Evaluation of CoQ10-SPGS Nanomicelles. Photostability tests were conducted with an YP-250GSP drug stability test chamber (Shanghai Maijie, Shanghai, China). Ten milliliters of the CoQ10-SPGS-2K or CoQ10-SPGS-5K nanomicelles and control solution was separately placed in transparent glass vessels and irradiated with eight TLD 15W/54−765 fluorescent tubes (Philips, Amsterdam, The Netherlands) at a distance of 25 cm, a temperature of 25 °C, and a radiation intensity of up to 4500 ± 500 Lx. Upon completion of irradiation tests, the remaining CoQ10 in the tested samples was determined by UV−vis spectrophotometry and HPLC. Differential Scanning Calorimetry and X-ray Diffraction. A DSC85le differential scanning calorimeter (Mettler Toledo, Zurich, Switzerland) was used to evaluate the thermal decomposition behavior of the CoQ10-SPGS-2K and CoQ10-SPGS-5K micelles at a heating rate of 10 °C/min in the temperature range of 10−80 °C. X-ray diffraction (XRD) patterns for the samples were recorded using a D8 Advance Xray diffractometer (Bruker, Karlsruhe, Germany) at a voltage of 40 kV and a current of 25 mA. The scanned angle was set as 10°≤ 2θ ≤ 80° at a step size of 0.04°. In Vitro Cytotoxicity of SPGS Micelles. The in vitro cytotoxicity of micelles was evaluated by the MTT assay on human L02 hepatocytes (HL-7702) and human liver carcinoma cells (HepG-2). Briefly, the cells in 100 μL of medium containing 10% fetal bovine serum (FBS) were seeded onto a 96-well plate at a density of 4 × 104 cells/well and incubated for 24 h (37 °C, 5% CO2). Then the medium of each well was replaced with 100 μL of the fresh medium supplemented with 10% FBS containing various concentrations of micelles and incubated for 48 h (37 °C, 5% CO2), followed by 100 μL of MTT solution (0.5 mg/mL). The cells were incubated for additional 4 h, and then 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the resulting purple formazan crystals. The optical densities at 570 nm were measured with a microplate reader. The cells cultured in medium containing 10% FBS alone (without SPGS micelles) were used as controls. Pharmacokinetic Study. Before experiments, Sprague−Dawley rats were randomly divided into three groups and fasted for 12 h except for free access to water. CoQ10-SPGS-2K, CoQ10-SPGS-5K, and CoQ10-suspension (CoQ10-suspension was prepared by uniformly mixing CoQ10 drug with 0.5% sodium carboxymethyl cellulose solution), with the same concentration of CoQ10, were given to the rats through intragastric administration at a dose of 65 mg/kg. Blood samples were collected from the retro-orbital plexus under mild

separately added into ten vessels. The resultant solutions of pyrene in acetone were left in the dark and dried in air. Then 5 mL of the SPGS micelle dispersions with different polymer concentration was added into each vessel (the concentration of the pyrene solution was fixed at 6.0 × 10−7 mol/L, and that of the polymer micelle was kept in the range from 4.504 × 10−7 mg/mL to 4.504 × 10−1 mg/mL). The fluorescence spectra were recorded with an F-4600 fluorescence spectrometer (Hitachi, Japan) under an excitation wavelength of 335 nm, with the fluorescence emissions at 373 and 384 being monitored. The CMC was estimated from the extrapolated cross-point of the fluorescence emission curves (the intensity ratio I384/I373 at low and high concentration regions was considered). Formation of CoQ10-Loaded SPGS Micelles. CoQ10-loaded micelles were prepared by the solvent evaporation method. Briefly, SPGS-2K or SPGS-5K (40 mg) and CoQ10 (40 mg) were dissolved in 2 mL of acetone. The resultant solution was added dropwise into 40 mL of pure water under room temperature magnetic stirring, followed by evaporation of acetone to afford CoQ10-loaded SPGS micelles. The resultant CoQ10-loaded SPGS micelle dispersions were filtered through a 0.22 μm syringe filter to remove undissolved CoQ10 and then sealed and stored in refrigerator. The drug loading content (DLC) of CoQ10 by SPGS micelles was determined by high performance liquid chromatography (HPLC). Namely, 5 mL of the CoQ10-loaded SPGS micelle dispersions was freeze-dried, and the residue was dissolved in ethanol. The amount of CoQ10 was determined by HPLC with an LC-20AT pump and SPD20A UV−vis detector (Shimadzu, Kyoto, Japan). The HPLC measurements were conducted at a wavelength of 275 nm, and the column used was a 250 mm × 4.6 mm i.d., 5 μm, Hypersil BDS C18, with a 10 mm × 4.6 mm i.d. guard column of the same material (Thermo Fisher Scientific, Waltham, MA). The mobile phase of methanol/ethanol (40:60, v/v) at a flow rate of 1.0 mL/min was adopted. By using a calibration curve obtained from CoQ10/ethanol solutions with different CoQ10 concentrations, the DLC value (%) was determined as the ratio of the weight of loaded CoQ10 drug to the total weight of the loaded drug and SPGS polymer. For convenience, CoQ10-SPGS-2K and CoQ10-SPGS-5K are referred to as CoQ10loaded SPGS-2K and SPGS-5K micelles, respectively. The morphologies of CoQ10-micelles are examined using a JEM-100CX II transmission electron microscope (TEM) (JEOL, Tokyo, Japan) at an accelerating voltage of 100 kV. 3362

DOI: 10.1021/acs.jafc.7b00165 J. Agric. Food Chem. 2017, 65, 3360−3367

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Journal of Agricultural and Food Chemistry anesthesia with ether at intervals of 20, 40, 70, 120, 240, 360, 480, 600, 720, and 1440 min and placed in heparinized tubes. The blood samples were centrifuged at 4000 rpm for 10 min to afford 200 μL of plasma. All the plasma samples were stored at −20 °C before further treatment. In the processing of biological samples, 35 μL of vitamin K1 (6.40 μg/mL) as an internal standard was added into 200 μL of plasma and vortex mixed. Then 400 μL of methanol was added to precipitate the protein, and 1 mL of n-hexane was added to extract the CoQ10 under 3 min of vortex. The remaining CoQ10 solution in hexane was centrifuged at 5000 rpm for 10 min, and then the hexane layer was collected in a clean glass tube. The residual solution was supplemented with 1 mL of hexane to repeatedly extract the remnant CoQ10. The hexane layers were finally evaporated under a nitrogen stream at 40 °C. The residue was collected and reconstituted in 100 μL of acetonitrile, and 20 μL of the resulting solution was analyzed by HPLC. The average plasma concentration versus time curves were plotted to time to reveal the different absorption profiles of CoQ10loaded SPGS formulations. Pharmacokinetic parameters were conducted with DAS 2.0 software. Cvmax (Maximum concentration) and Tmax (time needed for achieving the maximum concentration) values were calculated from actual CoQ10 plasma concentrations. AUC0−24h (area under the blood concentration vs time curve from t = 0−24 h after administration) was estimated with the linear trapezoidal rule.

bears a longer PEG chain than SPGS-2K. Therefore, SPGS-5K has a slightly higher CMC than SPGS-2K, similar to that reported reported elsewhere on amphiphilic block copolymers.42 The CMC value of TPGS is reported to be 0.02% (w/ w; approximately 200 μg/mL), and it is about 41-fold as much as that of SPGS.23 The SPGS micelles exhibit superior stability, which could be the reason that the core-forming solanesol has a much higher molecular weight (631.1 Da) than vitamin E (430.7 Da).39,41,42 Preparation and Stability of CoQ10-Loaded Micelles. CoQ10 exhibits an extremely low aqueous solubility (0.7 ng/mL at 37 °C) due to its large molecular weight (863 Da) and slow dissolution rate of CoQ10 associated with its “grease ball” nature.43 As shown in Figure 3A, the yellow powder of CoQ10

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RESULTS AND DISCUSSION Synthesis of SPGS and Formation of Their Micelles. The synthetic route of SPGS involves two steps of reactions, as shown in Figure 1. In the first step, monosolanesol solanesyl succinate (MSS) is obtained by the reaction between succinic anhydride and solanesol in the presence of TEA. As shown by the 1H NMR spectrum in Figure 2A, δ 2.65 corresponds to the methylene proton of methylene of succinic acid (−OOCCH2CH2COO−), and δ 4.61 belongs to the esterbonded methylene protons (−COOCH2−),38 which confirm that MSS was successfully synthesized. In the second step, SPGS is obtained by the esterification of MSS and mPEG under DCC/DMAP catalysis. When mPEG with different molecular weights of 2000 and 5000 Da is used, amphiphilic SPGS-2K and SPGS-5K with the same hydrophobic solanesyl segment and different hydrophilic segments (mPEG of 2000 and 5000 Da) are obtained. As shown in Figure 2B and C, the chemical shift values, δ 3.70 and δ 3.40, are ascribed to the characteristic protons of mPEG,39 and δ 4.25 and δ 4.61 are assigned to the protons of methylene adjacent to ester-bonded mPEG and solanesyl segments, respectively. The other characteristic signals correspond to the solanesol remnant of SPGS-2K and SPGS-5K. The micelles of amphiphilic SPGS were prepared by a solvent evaporation method. The average hydrodynamic diameters of the SPGS-2K and SPGS-5K micelles, determined by DLS, are 103 and 108 nm, respectively They show no obvious statistical difference in size, although they have mPEG segments with different lengths as the shells.39 The CMC value of a surfactant refers to the structural stability of its micelle under a diluted condition in vivo.40 In general, a low CMC is critical for enhanced bioavailability of the drug encapsulated in the micelles. Although both hydrophilic and hydrophobic blocks influence the CMC value, the hydrophobic block plays a more crucial role.41 The CMC values of the PEGylated solanesols SPGS-2K and SPGS5K are determined to be 5.36 μg/mL and 5.89 μg/mL, respectively, with pyrene as a probe. SPGS-2K and SPGS-5K exhibit the same hydrophobic solanesyl parts, but SPGS-5K

Figure 3. Photograph images of aqueous suspensions of (A) CoQ10, (B) CoQ10-SPGS-2K, and (C) CoQ10-SPGS-5K. TEM images of (D) CoQ10-SPGS2K and (E) CoQ10-SPGS5K.

was not dissolved and floats atop water at room temperature. After the encapsulation by SPGS, CoQ10 is dispersed in water to form homogeneous yellow solutions (Figure 3B,C). The CoQ10-loaded SPGS solutions show a Tyndall effect upon laser irradiation of laser, which indicates that the CoQ10-loaded SPGS micelles are of nanosize, as evidenced by relevant DLS results. The CoQ10-SPGS-2K and CoQ10-SPGS-5K micelles have an average diameter of 180 and 186 nm. Also, the CoQ10SPGS-2K and CoQ10-SPGS-5K micelles have much larger size than corresponding SPGS-2K and SPGS-5K micelles (103 and 108 nm), which is due to the incorporation of a large amount of CoQ10 into the core of SPGS micelles.44 Transmission electronic microscopy (TEM) images (Figure 3D,E) were taken to investigate morphology and size of micelles. Spherical shapes were investigated, and the sizes by TEM were consistent with that of DLS measurements. The drug loading content of CoQ10 into SPGS-2K and SPGS-5K micelles is about 39% and 49%, respectively. Sikoraka et al.45 reported a water-soluble noncovalent complex of CoQ10-PEGylated PTS with a molar ratio of 1:2.45 The DLC value of the CoQ10-PEGylated PTS is reported to be 23.2%, lower than that of CoQ10-SPGS-2K and 3363

DOI: 10.1021/acs.jafc.7b00165 J. Agric. Food Chem. 2017, 65, 3360−3367

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Journal of Agricultural and Food Chemistry CoQ10-SPGS-5K micelles, which means that the SPGS micelles exhibit a higher solubilization efficiency toward CoQ10 than the PTS micelle. XRD and DSC Analyses. The crystallinity of the encapsulated drug has effects on the micelle stability, drug release profile, and bioavailability of a drug delivery system.45 The XRD spectra of CoQ10 powder, blank SPGS-5K, the mixture of CoQ10 and SPGS-5K, and CoQ10-loaded micelles were investigated. The distinct sharp and intense peak of CoQ10 powder at 2θ = 18° corresponds to its high crystallinity.8 Blank SPGS-5K shows two sharp peaks also around 2θ = 18°. As a result, the physically mixed CoQ10/SPGS-5K shows overlapped XRD peaks. Differing from CoQ10, the CoQ10SPGS-2K and CoQ10-SPGS-5K micelle formulations show much weaker XRD peaks, which indicate that the CoQ10 drug exhibits a significantly reduced crystallinity in the SPGS-2K and SPGS-5K micelles. Relevant DSC data further elucidate the crystallinity of various samples. CoQ10 alone shows a sharp endothermic peak at 51.2 °C, and it is ascribed to the melting of CoQ10.1,14 The SPGS-5K alone displays a distinct endothermic peak at 56.2 °C, and it is attributed to the melting of mPEG (MW: 5000 Da). The physical mixture of CoQ10/SPGS-5K shows two melting peaks attributed to CoQ10 (50.4 °C) and mPEG 56.8 °C. The CoQ10-SPGS-2K and CoQ10-SPGS-5K micelle formulations show CoQ10-related peaks at lowered temperatures of 48.0 and 47.2 °C, respectively, and these CoQ10-related peaks correspond to the crystallization of CoQ10 in the micelles with a high drug loading content (more than 39% DLC).46 Such a decrease in the melting point of the loaded CoQ10 in CoQ10-SPGS-2K and CoQ10-SPGS-5K micelle formulations implies that unique molecular interactions take place between CoQ10 and noncrystalline solanesol core of the micelles. In other words, the encapsulation of a large amount of amorphous CoQ10 by SPGS micelles leads to a decrease in the melting point of the loaded CoQ10, as evidenced by corresponding XRD data. Colloidal Storage Stability and Dilution Stability. The change in the size of CoQ10-loaded SPGS micelle formulations during the storage of up to one month at the temperature of 4 and 25 °C was monitored by DLS. The particle sizes of the CoQ10-SPGS-2K and CoQ10-SPGS-5K micelle formulations rise slightly after the storage at 25 °C, and they remain nearly unchanged after the storage at 4 °C. This well corresponds to the excellent kinetic stability of the CoQ10-loaded SPGS micelle formulations subjected to visual observation.20 Furthermore, when CoQ10-SPGS-2K and CoQ10-SPGS-5K micelle solutions are diluted 1000-fold with water (a micelle concentration of 2.0 mg/mL), they undergo very slight changes in sizes, which also indicate that they exhibit excellent thermodynamic stability. It should be noted that the concentrations of the two solutions fell below the critical micelle concentration of SPGS-2K and SPGS-5K when the solutions were 1000-fold diluted. It could be that the increased interaction between CoQ10 and hydrophobic core of the micelles improved the stability of the CoQ10-SPGS-2K and CoQ10-SPGS-5K. Photostability of CoQ10 in CoQ10-SPGS Micelles. The exposure of CoQ10 solution to sunlight often yields superoxide and singlet oxygen, which can act as agents to accelerate the photodegradation of CoQ10.17 The photoprotection ability of PEGylated solanesol toward CoQ10 under simulated sunlight irradiation was assessed. Figure 4 shows the sunlight induced degradation of CoQ10 or SPGS-encapsulated CoQ10 at 25 °C as a function of irradiation time. After 24 h of exposure to the

Figure 4. Photodegradation of CoQ10 samples upon exposure to simulated sunlight.

simulated sunlight without the protection of the SPGS micelles, CoQ10 in water and ethanol is severely degraded by a rate of 71.8% or 45.7%. However, the SPGS-encapsulated CoQ10 undergoes significantly reduced degradation (to about 11.2%) under the same irradiation condition. This means that the encapsulation of CoQ10 by SPGS micelles contributes to significantly enhancing its photostability in both water and ethanol solution, which is possibly because the SPGS micelles as the carriers containing double bonds are able to absorb UV radiation. Namely, as the sacrificial protector of the SPGS micelle-encapsulated CoQ10, the solanesol core can not only well dissolve CoQ10, but also effectively absorb UV radiation and scavenge lipid free radicals.37 As a result, the photostability of the loaded CoQ10 is enhanced due to the presence of the SPGS micelles that function as the barrier to prevent the encapsulated CoQ10 drug from contact with UV rays, oxygen, and water.14,47,48 Cytotoxicity of the Blank Micelles. The toxicities of the blank SPGS micelles were tested in human L02 hepatocytes (HL-7702) and human liver carcinoma cells (HepG-2) by MTT assay. As shown in Figure 5, the cell viabilities of HepG-2

Figure 5. Cytotoxicity of SPGS micelles incubated with HepG-2 cells and HL-7702 cells for 48 h. 3364

DOI: 10.1021/acs.jafc.7b00165 J. Agric. Food Chem. 2017, 65, 3360−3367

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Journal of Agricultural and Food Chemistry

administration of CoQ10-SPGS-2K or CoQ10-SPGS-5K micelle formulations provides significantly increased Cmax and AUC0−24h values of 0.74 ± 0.07 mg/L and 379.33 ± 45.09 (mg min)/L or 0.55 ± 0.06 mg/L and 333.29 ± 97.04 (mg min)/L. However, crystalline CoQ10 and CoQ10-SPGS micelle formulations show insignificant difference in Tmax. Compared with crystalline CoQ10, CoQ10-SPGS achieved more than three-fold enhancement in the oral bioavailability. It was indicated that oral absorption of lipophilic CoQ10 was greatly enhanced by the self-assembled nanocarriers, which could be attributed to increased aqueous solubility of CoQ10. Nanoparticles have a strong muco-adhesive property.49 Long-lasting absorption of CoQ10 micelles in the gastrointestinal tract might result in the prolonged systemic exposure in CoQ10-SPGS, which might be part of the reason for the satisfactory biopharmaceutical properties of CoQ10-SPGS.

and HL-7702 are above 80% after 48 h of incubation in SPGS2K and SPGS-5K blank micelles with a concentration of 0.36 g/ L. The calculated half maximal inhibitory concentration (IC50) values for SPGS blank micelles were tested in HepG-2 cells and HL-7702 cells lines. The SPGS-2K micelle provides IC50 values of 0.532 g/L and 1.041 g/L for HepG-2 cells and HL-7702 cells, respectively. The SPGS-5K micelle exhibits IC50 values of 0.638 g/L and 1.362 g/L for HepG-2 cells and HL-7702 cells, respectively. These results reveal that both SPGS-2K and SPGS-5K blank micelles are nontoxic and biocompatible. Pharmacokinetic Behavior in Rats. In vivo pharmacokinetic study was conducted to investigate the possible improvement in the bioavailability of the SPGS micelleencapsulated CoQ10. Figure 6 shows the mean plasma

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00165. Table and figures of properties of drug loaded micelles (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: 86-371-23880680. Fax: 86-371-23880680. ORCID

Shiyong Song: 0000-0003-2208-608X

Figure 6. Plasma concentration−time profiles of free CoQ10, CoQ10SPGS-2K, and CoQ10-SPGS-5K after oral administration to SD rats at 65 mg/kg dose. Each data point is expressed as the average of six repeat measurements (n = 6).

Funding

This work was supported by the National Natural Science Foundation of China (NSFC 51375142), Research Fund for Excellent Young College Teachers of Henan Province, and a Key Project Funded by the Education Department of Henan Province.

concentration−time profiles of the SD rats after the separate oral administration of crystalline CoQ10 suspension as well as CoQ10-SPGS-2K and CoQ10-SPGS-5K micelle formulations at a single dose of 65 mg/kg. Corresponding pharmacokinetic parameters of CoQ10 are summarized in Table 1. After oral

Notes

The authors declare no competing financial interest.

■

Table 1. Pharmacokinetic Parameters of CoQ10 after Administration of CoQ10 Samplesa

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pharmacokinetic parameters samples

Tmax (min)

Cmax (mg/L)

AUC0−24h ((mg min)/L)

CoQ10 crystalline CoQ10-SPGS-2K CoQ10-SPGS-5K

210 ± 120.56 150 ± 60.0 210 ± 60.00

0.21 ± 0.02 0.74 ± 0.07 0.55 ± 0.06

110.09 ± 15.82 379.33 ± 45.09 333.29 ± 97.04

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a Cmax, maximum concentration; Tmax, time needed for achieving the maximum concentration; AUC0−24h, area under the blood concentration vs time curve from t = 0−24 h after administration. Data of six repeat measurements with standard deviations (±) are listed.

administration of the crystalline CoQ10, the CoQ10 level in the plasma is calculated to be Cmax = 0.21 ± 0.02 mg/L and AUC0−24h = 110.09 ± 15.82 (mg min)/L. Onoue et al.17 reported similar results, and they attributed the low CoQ10 level in the plasma to the poor solubility of crystalline CoQ10 in aqueous solution. Different from the above-mentioned, the oral 3365

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