Article pubs.acs.org/JAFC
Preparation and Characterization of Mucoadhesive Nanoparticles for Enhancing Cellular Uptake of Coenzyme Q10 Ji-Soo Lee, Ji Woon Suh, Eun Suh Kim, and Hyeon Gyu Lee* Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea ABSTRACT: The mucoadhesive nanoparticles (NPs) for oral delivery of coenzyme Q10 (CoQ10) were prepared using natural mucoadhesive polysaccharides, chitosan (CS), and dextran sulfate sodium salt (DS) in order to improve the solubility, cellular uptake, and thermo- and photostability of CoQ10. CoQ10-loaded NPs were prepared in the range of 340−450 nm with an entrapment efficiency of 60−98%. The mucoadhesiveness and cellular uptake of NPs were evaluated by measuring the amount of mucin adsorbed on NPs and CoQ10 absorbed in Caco-2 cells, respectively. CS/DS NPs had higher mucoadhesive strength than CS/sodium triphosphate pentabasic NPs (control group). Moreover, the solubility, cellular uptake, thermo- and photostability of CS/DS NPs were significantly improved compared with non-nanoencapsulated free CoQ10. Particularly, CS/DS NPs prepared with 0.5 mg/mL of CS and DS produced the highest mucoadhesiveness, solubility, cellular uptake, and cellular antioxidant activity. Thus, mucoadhesive CS/DS NPs may be an effective oral delivery platform for improving bioavailability of CoQ10. KEYWORDS: coenzyme Q10, nanoencapsulation, mucoadhesive, stability, cellular uptake
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INTRODUCTION Coenzyme Q10 (CoQ10) is a vitamin-like substance found in cell membranes throughout the body, primarily in mitochondrial membranes. In the mitochondria, CoQ10 is a component of the electron transport chain, where it serves a key role as an electron carrier and proton translocator in aerobic cellular respiration, generating energy in the form of adenosine triphosphate.1 CoQ10 scavenges free radicals, inhibits lipid peroxidation as an antioxidant, and has also been found to be beneficial against cardiovascular disorders, migraine headache, and neurodegenerative disease.2 Unfortunately, CoQ10 is poorly absorbed from the gastrointestinal tract due to low solubility, low thermal- and photostability, and high molecular weight.3 Furthermore, P-glycoprotein (P-gp) mediated efflux transport of CoQ10 limits absorption of CoQ10.4 Various approaches, including the use of oily solutions, solid dispersion systems, liposomes, cyclodextrin complexes, selfemulsified drug delivery systems, and nanosuspensions have been taken to overcome the drawbacks of CoQ10.5−9 Most of these studies more focused on the physicochemical properties of formulations than the cellular uptake of CoQ10, and only some studies used materials safe for food. Thus, there is a need for an efficient method utilizing nontoxic and biodegradable materials to improve the solubility, stability, and absorption of CoQ10.10 Encapsulation is defined as the technology of entrapping pure materials or a mixture that can release their contents under specific circumstances.11 The main objective of encapusulation is to protect bioactive components from adverse environment conditions such as oxidation, pH, light, and enzyme degradation. Nanoencapsulation involves the formation of particles with diameters between 1 and 1 000 nm that are loaded with active ingredients.12 Compared to microsized particles, nanoparticles (NPs) can provide more surface area and enhance solubility and bioavailability.13 In addition, the high ratio of surface area to volume of nanoencapsulation © 2017 American Chemical Society
systems can be used to establish an adhesive interaction between NPs and mucus or mucin.14,15 Mucoadhesion is defined as the state that occurs when two materials are in close contact and remain together for extended periods of time due to interfacial forces and is especially prevalent in mucosal membranes. Mucoadhesive systems have been applied as a way of improving bioavailability by prolonging residence time in mucosa.16 This prolonged residence time results from the fact that mucoadhesive formulations enable intimate contact with the absorption surface of the mucous membrane due to interactions with each other. In addition, it allows diffusion and penetration of mucoadhesive materials to the mucus layer, contributing toward improved absorption.17 In order to utilize mucoadhesive properties in a drug delivery system, it is necessary to use specific polymers with mucoadhesive properties. The adhesion between biopolymers and mucus surface is due to surface interactions including ionic bonding, covalent bonding, hydrophobic interactions, and physical entanglement.18 Both chitosan (CS) and dextran sulfate sodium salt (DS) have been reported as mucoadhesive biopolymers. Specifically, the positive charge of CS allows it to bind to negatively charged mucus surfaces through ionic bonding. Müller et al.19 demonstrated that the nanosuspensions modified with CS increased retention time in the gastrointestinal tract. In addition, sulfate functional groups of DS have been shown to form strong hydrogen bonding interactions with mucin.15,20 The mucoadhesive properties of CS/DS NPs were confirmed on both mucin and buccal cells in our previous study.21 Thus, nanoencapsulation using CS and DS may be Received: Revised: Accepted: Published: 8930
July 18, 2017 September 19, 2017 September 21, 2017 September 21, 2017 DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
Article
Journal of Agricultural and Food Chemistry
methanol (90:10% v/v). Separation was carried out using a Capcell Pak C18 column (250 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL/ min with UV detection at 275 nm. EE was determined by analyzing the amount of non-nanoencapsulated free CoQ10 in supernatant after ultracentrifuging the CoQ10-loaded NPs at 30 000g for 30 min (Optima TL ultracentrifuge, Beckman, Fullerton, CA). EE was calculated using the following eq 1:
suitable for improving cellular uptake and mucoadhesion of CoQ10. It has been reported that improved oral drug bioavailability can be promoted by administration of efflux pump inhibitors. P-gp is a membrane transporter that actively pumps xenobiotics out of the cells by contacting with the membrane both inside and outside of the cell and spanning the cell membrane.22,23 The absorption of CoQ10 can be improved by inhibition of Pgp mediated efflux in a cell monolayer.24 Various P-gp inhibitors have been studied such as antibiotics, herbal constituents, and polymer formulations. Especially, polymer formulations including nanoparticulate systems allow drugs to bypass efflux pump transport and avoid recognition by P-gp, allowing delivery inside the cell.25 Therefore, it is assumed that application of nanoencapsulation as a P-gp inhibitor can be lead to improved absorption of CoQ10. The aim of this study was to prepare mucoadhesive NPs using natural biopolymers for enhancing the cellular uptake of CoQ10. For this purpose, CoQ10-loaded NPs were prepared using CS and DS and their physicochemical properties were characterized including particle size, polydispersity index (PDI), derived count rate, and encapsulation efficiency (EE). The effect of CoQ10 nanoencapsulation using CS and DS on its mucoadhesive ability, solubility, thermostability, photostability, and cellular uptake were evaluated.
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EE (%) =
total amount of CoQ10 − free amount of CoQ10 in supernatant total amount of CoQ10 × 100
(1)
Mucoadhesive Properties of NPs. The mucoadhesive properties of NPs to mucin were evaluated as previously described with modification.2 Briefly, 0.6 mL of a mucin solution (0.5 mg/mL) was mixed with 0.6 mL of a nanosuspension and incubated at 37 °C in a shaking water bath for 1 h. Afte centrifugation at 10 000 rpm for 5 min, the supernatant was collected and the amount of free mucin was measured by the Bradford protein assay. For the protein assay, the supernatant was incubated with the Bradford reagent for 5 min, and the absorbance was measured at 595 nm with a spectrophotometer (Biomate 3S, Thermo Scientific, Waltham, MA). The amount of mucin adsorbed to the NPs was calculated as the difference between the total amount of mucin added and the residual mucin in the supernatant. The concentration of mucin was calculated from a standard curve. Solubility of CoQ10. Solubility analysis for free CoQ10 and CoQ10-loaded NPs were determined using the method described by Sun et al.28 with modification. To quantify the solubilized CoQ10, an extraction procedure was applied for both free CoQ10 and NP suspensions. First, 200 μL of methanol and 600 μL of hexane were added to 100 μL of each sample and mixed thoroughly with a vortex mixer for 3 min. Then, the mixture was centrifuged at 10 000 rpm for 10 min (Combi-408, Hanil Science Ins., Incheon, Korea), and 500 μL of supernatant was removed. This extraction procedure was repeated twice. The total supernatant was collected and dried using a speed vacuum concentrator (Ecospin 3180C, BioTron, Daejeon, Korea). The residue was reconstituted in 300 μL of ethanol and the resulting suspensions were filtered through a polytetrafluoroethylene membrane filter with a pore size of 0.45 μm (Xiboshil, Tianjin Fuji Tech Co., Tianjin, China). Filtered solutions containing CoQ10 were measured by HPLC. Stability of CoQ10. For thermostability analysis, samples of CoQ10-loaded CS/DS NPs, CS/TPP NPs, and free CoQ10 were prepared and maintained at 60 °C in a water bath. The CoQ10 content of each sample was investigated at 24 h intervals for up to 4 days using a validated HPLC method and the extraction procedure described above.28,29 For photostability analysis, samples of CoQ10-loaded CS/DS NPs, CS/TPP NPs, and free CoQ10 were prepared and irradiated for 8 h at 25 °C with a 254 nm UV lamp (Vilber Lourmat, Torcy, France).30,31 The distance between the sample and UV light was 15 cm. Samples were withdrawn at 1 h intervals and the CoQ10 content was determined using the extraction procedure described above and a validated HPLC method. Transcellular Transport Study. For transcellular transport analysis, monolayer cultures grown in a 12-well transwell were used. Cell monolayers were fed fresh medium every other day before performing transport experiments. Transepithelial electrical resistance (TEER) was used to monitor the integrity of monolayers. Only monolayers exceeding 400 Ω cm2 were used. HBSS-HEPES buffer was used as the incubation medium for the transcellular transport study. After removal of the growth medium from both the apical and basolateral sides of the monolayer, the cells were preincubated at 37 °C for 10 min with HBSS-HEPES buffer (12-well; 1.5 mL of basolateral and 0.5 mL of apical). After removal of the incubation medium, medium containing CoQ10 was added to the outside chamber. For efflux experiments, incubation medium containing nanosuspensions was added to the inside chamber. In all experiments,
MATERIALS AND METHODS
Materials. CoQ10 and CS (water-soluble, molecular weight (MW) of 1 000−3 000, 24 cps, 95% deacetylated) were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Kittolife Co. (Seoul, Korea), respectively. DS (MW, 15 000), sodium triphosphate pentabasic (TPP), Coumarin 6 (C6), and mucin (extracted from porcine stomach, type III) were purchased from Sigma-Aldrich Co. (St Louis, MO). The colon carcinoma (Caco-2) and human hepatocellular carcinoma (HepG2) cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD) and Korean Cell Line Bank (Seoul, Korea), respectively. Dulbecco’s Modified Eagle’s culture medium (DMEM), nonessential amino acid (NEAA), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), and 0.25% trypsin-EDTA were purchased from Gibco Invitrogen Co. (Grand Island, NY). Preparation of NPs. Two types of CoQ10-loaded NPs, CS/DS and CS/TPP NPs, were prepared using CS, DS, and TPP.26 First, CS was dissolved in distilled water at a concentration of 1.25 mg/mL. Next, CoQ10 was dissolved in ethanol at a concentration of 2.0 mg/ mL, and 0.75 mL of the CoQ10 solution was mixed with 3 mL of the aqueous CS solution. CS/DS NPs were prepared by adding 3 mL of the DS solution (0.0125 mg/mL, 0.0375 mg/mL, or 0.0625 mg/mL) to the CS and CoQ10 mixture for 10 min under magnetic stirring (1 000 rpm). The formation of CS/TPP NPs was initiated by mixing 3 mL of the TPP solution (0.0125 mg/mL) with the already mixed CS/ CoQ10 solution. Physical Characteristics of NPs. The particle size and PDI of NPs were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, U.K.). Measurements were taken using multiple narrow modes at 25 ± 1 °C. Transmission electron microscopy (TEM, JEM 2100F, JEOL, Tokyo, Japan) was used to analyze the morphology of NPs. Samples for TEM analysis were placed on a 200-mesh carbon-coated copper grid and stained for 30 min with a 2% phosphotungstic acid solution (Sigma-Aldrich Co.). After staining, samples were air-dried at 37 °C before loading onto the microscope. EE of NPs. The EE of the CoQ10-loaded NPs was determined using a reverse-phase high performance liquid chromatography (HPLC) system consisting a 486 tunable absorbance detector, two 515 pumps, and a manual injector with a 50 μL loop (all from Waters Corp., Milford, MA).27 The mobile phase was a mixture of ethanol and 8931
DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
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Journal of Agricultural and Food Chemistry monolayers were incubated for 120 min at 37 °C. For transport measurements, aliquots of the incubation solution were withdrawn from the apical side at specified times and analyzed immediately.24,32 The apparent permeability coefficient (Papp) was calculated using the following eq 2:
Papp = dQ /dt × 1/(A × Co)
included triplicate controls (containing cells treated with DCFH-DA and HBSS with ABAP) and blank wells (containing cells treated with DCFH-DA and HBSS without ABAP). The CAA value was calculated using the following eq 4:36
CAA value = 100 −
(2)
where dQ/dt is the linear appearance rate of mass in the receiver solution, A is the area of the cell monolayer (1.13 cm2), and Co is the initial substrate concentration. Cellular Uptake Study. Qualitative Study (Confocal Laser Scanning Microscopy). Caco-2 cells (4 × 105 cells/well) were seeded on glass-bottom dishes and allowed to adhere at 37 °C until reaching 50−70% confluency. The cells were then exposed to free C6, C6loaded CS/TPP NPs, or CS/DS NPs (equivalent to 1 μg/mL C6) and incubated for 2 h. After incubation, the cell culture medium was aspirated and cells were rinsed with phosphate buffered saline (PBS). Next, the cells were fixed with 70% ethanol in PBS (pH 7.4) for 15 min at room temperature. The cells were then washed twice with cold PBS, and nuclei were stained with propidium iodide (PI) for 30 min. The coverslips were then isolated carefully from wells and mounted on slide glasses. The cellular uptake of NPs was observed using confocal laser scanning microscopy (CLSM) (TCS SP5, Leica Co., Ltd., Wetzlar, Germany) (excitation at 420 nm, emission at 505 nm). The fluorescence intensity of sample was analyzed using confocal images, Leica confocal software (Leica Microsystems, Heidelberg, Germany), and ImageJ software (NIH, Bethesda, MD). Uptake Study in Caco-2 Cell Monolayers. Uptake experiments were performed as previously described with modification.33,34 Briefly, the uptake of CoQ10 was measured using monolayer cultures grown in 24-well transwells. The cell monolayers were fed fresh medium every second day before transport experiments. Cultures with a TEER greater than 350 Ω cm2 were used. After medium was removed from both the apical and basal chamber, 0.6 mL of medium was added to the basolateral side for preincubation at 37 °C for 10 min. After removal of the medium, 0.2 mL of CoQ10-loaded NPs was added to the apical chamber. The monolayers were incubated for 120 min at 37 °C. After incubation, cell monolayers were rapidly washed twice with 1 mL of ice-cold incubation medium. The cells were then suspended in 1 mL of methanol for extraction and sonicated to disrupt the cells. An extraction solution was used to determine the uptake of CoQ10 after centrifugation of the mixture (10 000 rpm, for 5 min). Antioxidant Activity Study. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay. DPPH radical scavenging capacity was determined using the Brand-Wiliams method with modifications.35 Briefly, a DPPH solution in anhydrous ethanol (100 μL, 0.5 mM) was added to each sample solution (100 μL). The reaction mixture was then incubated at 25 °C for 30 min in dark. The scavenging activity on DPPH free radical was determined by measuring the absorbance at 517 nm with a Synergy HT Multimicroplate reader (BioTek Instruments, Winooski, VT). The radical scavenging effect was calculated using the following eq 3:
∫ SA × 100 ∫ CA
(4)
where ∫ SA is the integrated area under the sample fluorescence versus time curve and ∫ CA is the integrated area from the control curve Statistical Analysis. All experiments were carried out in triplicate and expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with Duncan’s multiple range test. P-values below 0.05 were considered statistically significant. The SPSS, version 21.0 statistical package (SPSS Inc., Chicago, IL) was used for statistical analysis.
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RESULTS AND DISCUSSION Physical and Mucoadhesive Properties of CS/DS NPs. In order to select the appropriate concentration of DS (NP wall
Figure 1. Physical properties (A) and mucoadhesiveness (B) of CS/ DS nanoparticles with different DS concentrations. Different letters indicate significant difference (p < 0.05).
(Ac − (As − Ab)) × 100 Ac (3) where Ac is the absorbance of the control, As is the absorbance of the sample, and Ab is the absorbance of the blank. Cellular Antioxidant Activity (CAA) Assay. HepG2 cells were seeded into 96-well plates at a density of 6 × 104 cells per well. After incubation for 24 h, the growth medium was removed and the cells were washed with PBS. Triplicate wells were treated for 1 h with 100 μL of CoQ10 solution or CoQ10-loaded NPs plus 25 μM dichlorodihydro-fluorescein diacetate (DCFH-DA) dissolved in treatment medium. After incubation for 1 h, the wells were washed with 100 μL of PBS. Then, 600 μM 2,2′-azobis dihydrochloride (ABAP) was applied to the cells in 100 μL of HBSS, and the 96-well microplate was placed into a Synergy HT Multimicroplate reader (BioTek Instruments, Winooski, VT). Emission at 538 nm was measured with an excitation wavelength at 485 nm every 5 min for 1 h. Each plate DPPH radical scavenging effect (%) =
material), the CS/DS NPs were prepared with different concentrations of DS and their physical properties and mucoadhesiveness were investigated (Figure 1). As the dextran sulfate concentration increased, the particle size and PDI significantly increased. A PDI less than 0.3 may be associated with a monodisperse system, whereas a PDI greater than 0.3 might be indicative of heterogeneity.37 When the concentration of DS exceeded 0.4 mg/mL, the average size and PDI were higher than 500 nm and 0.4, respectively, resulting in an unstable particulate system. In addition, the amount of adsorbed mucin increased with increasing DS concentrations, although this trend was not observed when the concentration 8932
DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Properties of CoQ10-Loaded CS/DS and CS/TPP Nanoparticlesa polymer dextran sulfate
sodium triphosphate
nanoparticles
(mg/mL)
(mg/mL)
CS/DS NP1 CS/DS NP2 CS/DS NP3
0.1 0.3 0.5
CS/TPP NP
0.05
particle size
polydispersity index
derived count rate (kcps)
(%)
385.2 ± 19.4 ab 340.1 ± 7.3 b 447.1 ± 68.3 a
0.23 ± 0.0 b 0.18 ± 0.0 b 0.34 ± 0.2 a
169 224 ± 31 495 b 229 567 ± 50 154 a 219 438 ± 22 513 a,b
65.0 ± 4.6 b 92.9 ± 7.3 a 96.6 ± 1.5 a
358.4 ± 50.7 b
0.18 ± 0.0 b
188 267 ± 16 319 a,b
58.4 ± 4.5 b
(nm)
EE
a
Means with different letters are significantly different at p < 0.05. Concentrations of chitosan and CoQ10 are 0.5 and 0.2 mg/mL, respectively for all samples.
Figure 2. Transmission electron micrograph images of CS/DS nanoparticles.
Figure 3. Mucoadhesiveness of the non-nanoencapsulated and nanoencapsulated CoQ10. Different letters above the bars indicate a significant difference (p < 0.05).
Figure 4. Solubility of the non-nanoencapsulated and nanoencapsulated CoQ10. Different letters above the bars indicate a significant difference (p < 0.05).
of DS exceeded 0.5 mg/mL. The sulfate groups in DS appeared to have interacted strongly with the negatively charged mucin, causing higher mucoadhesion ability. According to electronic theory, there is an electrical double layer at the interface between the bioadhesive site and the glycoprotein chains of the mucus due to a transfer of electrons. Bioadhesion occurs because of an attraction across this electrical double layer.38 Batchelor et al.39 indicated that the retention of sulfate modified microparticles in esophageal tissue is greater than that of carboxylate particles. These results are expectable because increasing the dextran sulfate involved in particle formation increased particle size and the adhesive capacity of CS nanoparticles with mucin increased due to the mucoadhesive property of dextran sulfate. Physicochemical Properties of CoQ10-Loaded NPs. In order to investigate the physicochemical properties of CoQ10loaded mucoadhesive NPs, CoQ10-loaded CS/DS NPs (CS/
DS NP1, CS/DS NP2, and CS/DS NP3) were prepared using different concentrations of DS (0.1, 0.3, and 0.5 mg/mL) and CoQ10-loaded CS/TPP NPs were prepared as a control (Table 1). Physicochemical properties of NPs such as an average particle size, PDI, derived count rate (DCR), and EE were investigated. The size of CS/DS and CS/TPP NPs ranged from 340 to 447 nm. The effect of DS concentration was not clearly observed in the size of CoQ10-loaded CS/DS NPs. This result was similar to the description by Tiyaboonchai and Limpeanchob,40 who reported that particle size was not related to CS/DS ratios over the range of 1:6 to 1:2. The PDI of the CoQ10-loaded NPs in this study ranged from 0.2 to 0.3, which indicated that the NPs were homogeneous in the particle population (Table 1).37 DCR indicates the intensity of light scattered from particles and 8933
DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
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Figure 5. Stability of non-nanoencapsulated and nanoencapsulated CoQ10 at 60 °C in the absence of light (A) and under UV irradiation (λ = 254 nm) at 25 °C (B). Different letters indicate a significant difference (p < 0.05).
Figure 8. DPPH radical scavenging activities (A) and CAA values of free CoQ10 and CoQ10-loaded nanoparticles (B). Different letters above the bars indicate a significant difference (p < 0.05).
increases as the number or size of particles increases.41 Thus, preparation of CoQ10-loaded CS/DS NPs at a DS of 0.3 mg/ mL seemed to produce the largest amount of the smallest homogeneous NPs in suspensions from the results of its highest DCR values and smallest particle size and PDI. In addition, TEM examination of the morphology of CoQ10-loaded CS/DS NPs showed that NPs had a spherical morphology, low PDI, and an average size of 200−300 nm (Figure 2). The EE of the CoQ10-loaded CS/DS NPs significantly increased from 65 to 96% with an increase in DS from 0.1 to 0.5 mg/mL (Table 1). This result can be explained considering that the denser structure of CoQ10-loaded CS/DS NPs prepared with high DS concentration may reduce diffusional losses of CoQ10 from CS/DS NPs during preparation. In addition, the EE of the CoQ10-loaded CS/DS NPs was greater than that of CS/TPP NPs. This result suggested that the CS/ DS NPs could be used as effective carriers for CoQ10. Consistently, Cho et al.26 indicated that the presence of sufficient negative charges in DS enables high encapsulation of hydralazine during complexation with chitosan. Mucoadhesive Study of CoQ10-Loaded NPs. To evaluate the effect of CS/DS nanoencapsulation on mucoadhesive properties, the adsorption of mucin on free CoQ10 and CoQ10-loaded CS/DS and CS/TPP NPs was measured (Figure 3). As the concentration of DS increased, the amount of mucin adsorbed NPs increased significantly. These results indicated that CS/DS NPs prepared with high DS concentration exhibited a potent mucoadhesive property regardless of
Figure 6. Effect of CS/DS and CS/TPP nanoencapsulation on the transepithelial flux of CoQ10 by Caco-2 cell monolayers. Different letters above the bars indicate a significant difference (p < 0.05).
Figure 7. Quantitative uptake of non-nanoencapsulated and nanoencapsulated CoQ10 in Caco-2 cells. Different letters above the bars indicate a significant difference (p < 0.05).
8934
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encapsulation. Together, these results suggest that CS NPs are an effective carrier for stabilizing unstable CoQ10. Transcellular Transport Study of CoQ10-Loaded NPs. The effect of CS/DS and CS/TPP nanoencapsulation on the basal to apical transport of CoQ10 was investigated using Caco2 cells (Figure 6). In the presence of NPs, the basolateral to apical efflux transport of CoQ10 was reduced by almost 50%. In addition, there was no significant difference between CS/DS NPs and CS/TPP NPs. These results may suggest the possibility of using the NPs to effectively evade P-gp-mediated efflux. Nanoparticulate systems may inhibit efflux pumps by bypassing efflux transporters and P-gp. Such inhibited efflux could lead to enhanced delivery of CoQ10 to the cytoplasm and cell nucleus.32,47 Carreno-Gomez and Duncan48 used dextrans as efflux pump inhibitors for the oral delivery of drugs and showed that they could modify efflux pump activity. Moreover, in terms of safety, NPs using natural polymers have an advantage compared to other P-gp inhibitors such as anticancer drugs and antibiotics as well as synthetic compounds and molecules generated by parent-molecule optimization. Currently, the ideal P-gp inhibitor is considered to be a molecule that is nontoxic and does not have any pharmacological activity of its own.47 Cellular Uptake Study of CoQ10-Loaded NPs. Internalization of fluorescent CS/DS NPs by Caco-2 cells was visualized by CLSM. Images obtained from the fluorescein (FITC) channel showed the green fluorescence of the C6loaded CS/DS NPs, while images obtained with the PI channel showed red fluorescent cell nuclei. Merged FITC and PI images were used to determine nuclear or cytoplasmic localization of CS/DS NPs internalized by Caco-2 cells.49 The fluorescence intensity of free C6 and C6-loaded CS/DS NPs was quantified using ImageJ and the mean pixel intensities were 23.9 and 27.8, respectively. The difference between free C6 and C6-loaded CS/DS NPs was not clearly observed in qualitative cellular uptake studies (data not shown). As shown in a quantitative cellular uptake study, CS/DS NPs prepared with 0.5 mg/mL CS and DS appeared to be most effective in enhancing the cellular uptake of CoQ10, leading to a 3-fold improvement in uptake of CoQ10 (Figure 7). Moreover, the cellular uptake was significantly increased as the concentration of DS increased. There are several possible explanations for this observation. First, CS may have reversibly opened tight junctions between cells resulting in improved paracellular permeability.50 Consistent with this possibility, a number of studies have reported the use of CS as a permeation enhancer for facilitating drug absorption.49 Second, CS/DS NPs may have interacted with the cell monolayer through mucoadhesive abilities. Indeed, we observed excellent mucoadhesive properties of CS/DS NPs, and thus the improved mucoadhesion could have been due to enhanced cellular accumulation of CoQ10. Shao et al.2 also investigated the relationship between mucoadhesiveness of CS-coated layers and cellular uptake of CoQ10. Third, reduced P-gp mediated efflux of CoQ10 by CS/DS NPs may have enhanced cellular uptake of CoQ10. Itagaki et al.24 demonstrated that improved absorption of CoQ10 was achieved by intestinal P-gp inhibition. In addition, the improved solubility of CoQ10 by nanoencapsulation contributed to the enhanced cellular uptake of CoQ10. Lastly, CS/DS NPs were an effective alternative to the widely used CS/TPP NPs. Antioxidant Activity Study of CoQ10-Loaded NPs. To evaluate the formulation effect on antioxidant activity of free
the encapsulated core material used. In particular, the CoQ10loaded CS/DS NP3 prepared with 0.5 mg/mL of DS showed the highest mucoadhesive strength, which was greater than that of free CoQ and CS/TPP NPs, with approximately 3- and 30fold higher adsorption, respectively. Therefore, we assumed that CoQ10-loaded CS/DS NPs prepared with high DS concentration may have increased the residence time of CoQ10 in the intestines through their strong mucoadhesive property. Solubility Study of CoQ10-Loaded NPs. In order to investigate the effect of nanoencapsulation on the solubility of CoQ10, the solubility of non-nanoencapsulated free and nanoencapsulated CoQ10 were measured in 10% ethanol, which was ethanol concentration in the CoQ10-loaded NP suspensions (Figure 4). The solubility of CoQ10 increased by CS/DS nanoencapsulation. In addition, as the concentration of DS increased, the solubility of CoQ10 entrapped in CS/DS NPs significantly increased. The solubility of free CoQ10 ranged from 118 to 128 μg/mL, while that of CS/DS NP 3s prepared with 0.5 mg/mL of DS ranged from 140 to 164 μg/ mL. These results suggested that the improved wettability of NPs may have increased the solubility of CoQ10 by enhancing the surface area available for dissolution. Indeed, size reduction is one of the most important potential approaches to improve the bioavailability of hydrophobic drugs by increasing surface area and saturation solubility.12 However, the increased solubility (153 μg/mL) in this study was not superior to other studies using surfactant and synthetic polymer. The solubility of CoQ10 was enhanced to 346−500 μg/mL and 562 μg/mL by using poloxamer 188 and Labrasol, respectively.42,43 Nevertheless, our results are meaningful in terms of the use of biocompatible natural polymers, ease of the manufacturing procedure, and ability to produce the particles in an aqueous environment without the need for organic solvents or surfactant.40 Stability Study of CoQ10-Loaded NPs. To investigate the effect of nanoencapsulation on thermostability and photostability of CoQ10, the amount of CoQ10 after storage at 60 °C as a function of time was analyzed by HPLC as described above. Figure 5A shows the degradation profiles of CoQ10 after incubation for 4 days at 60 °C in the absence of light. While free CoQ10 exhibited the highest degree of degradation (about 70%), the stability of CoQ10 was significantly improved by nanoencapsulation, with 67−81% of CoQ10 remaining after 4 days. There was no clear difference among the thermostabilities of CoQ10-loaded CS NPs. This enhanced thermostability might have been due to the CS network structure, which can protect the entrapped substances such as CoQ10 by isolating them from the surrounding environment.30,44 Figure 5B shows the degradation profiles of CoQ10 during exposure time to UV illumination (254 nm) at 25 °C for 8 h. When CoQ10 is exposed to light, it gradually decomposes and its color changes from yellow to dark yellow. The amount of remaining non-nanoencapsulated free CoQ10 after 8 h was approximately 28%. However, the nanoencapsulated CoQ10 after 8 h was maintained at 47−50%. The difference among of CS NPs was not clearly observed in the photostabilities of CoQ10. The polymeric NPs may have acted as a barrier, preventing UV illumination from reaching the entrapped drug.45 Consistent with our observations, Kwon et al.46 reported that the photo and thermal resistance of CoQ10 incubated at 45 °C for 25 days is enhanced by nano8935
DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
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Journal of Agricultural and Food Chemistry
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and nanoencapsulated CoQ10, DPPH, and CAA assays were conducted (Figure 8). The DPPH assay has been widely used to evaluate the ability of compounds to act as hydrogen donors of free radical scavengers. CAA values in the CAA assay were calculated from data obtained from DCF fluorescence of cultured cells treated with antioxidant compounds.36 In the DPPH study, there was no significant difference between samples. However, in the intracellular antioxidant activity assay, the CAA value of CoQ10-loaded CS/DS NPs was significantly greater than that of free CoQ10. These results may be explained by the fact that the DPPH assay does not reflect the uptake, bioavailability, and distribution of antioxidant compounds.36 However, CAA may associated with the difference owing to mucoadhesion and cellular uptake ability between samples. According to the results of the CAA assay, CS/DS NPs exhibited a higher CAA value than both CS/TPP NPs and free CoQ10. These results were consistent with findings from the cellular uptake study and mucoadhesive study. Therefore, CS/DS nanoencapsulation is found to be a valuable technique in terms of improving the mucoadhesiveness, solubility, stability, cellular uptake, and antioxidant activity of CoQ10.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +82-2-2220-1202. Fax: +82-2-2281-8285. E-mail:
[email protected]. ORCID
Hyeon Gyu Lee: 0000-0002-9141-9469 Funding
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2014M3A7B4051898). Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.jafc.7b03300 J. Agric. Food Chem. 2017, 65, 8930−8937
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Journal of Agricultural and Food Chemistry
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