To improve the bioavailability of orally administered lipophilic coenzyme Q10 (CoQ10), we formulated a novel lipid-free nano-CoQ10 system stabilized by various surfactants. Nano-CoQ10s, composed of 2.5% (w/w) CoQ10, 1.67% (w/w) surfactant, and 41.67% (w/w) glycerol, were prepared by hot high-pressure homogenization. The resulting formulations were characterized by particle size, zeta potential, differential scanning calorimetry, and cryogenic transmission electron microscopy. We found that the mean particle size of all nano-CoQ10s ranged from
Coenzyme Q10 (CoQ10), an essential component of the mitochondrial respiratory chain, is found in the inner mitochondrial membrane of all living cells. It is an efficient antioxidant against free radicals and lipid peroxidation [
To improve the bioavailability of CoQ10, previously reported formulation strategies include an oil solution and suspension system [
Thus, the objective of the present study was to increase the solubility and improve the bioavailability of CoQ10 by developing and characterizing a novel nanoformulation with a higher CoQ10 relative to surfactant content, while minimizing the content of surfactant to avoid potential toxic clinical effects. We developed novel CoQ10 nanoformulations that included various surfactants but no other lipids by using the established hot high-pressure homogenization (HPH) method [
Soybean lecithin (SL, Epikuron 170V) was purchased from Cargill Texturizing Solutions Deutschland GmbH & Co. KG (Germany), and CoQ10 was purchased from Zhejiang Medicine Co. Ltd., Xinchang Pharmaceutical Factory (China). D-
A series of nano-CoQ10 formulations stabilized with different surfactants, such as TPGS, Cremophor RH40, SWA-10D, P-1670, Epikuron 170V, was prepared by hot HPH using a high-pressure homogenizer (model NS1001L, Niro Soavi, Italy) [
The CoQ10-suspension was prepared using a high-shear mixer. Briefly, 1 g Kollidon 30 was dissolved in 57.5 g glycerol aqueous solution (43.5%, w/w) at room temperature. Next, 1.5 g CoQ10 powder was dispensed into the resulting water solution to form a dispersed suspension at room temperature by using a high-shear mixer at 8000 rpm for 1 min.
The mean particle size and zeta potential value of the nano-CoQ10 were determined by means of dynamic light scattering using a Malvern Zetasize 2000 (Malvern Instruments, UK). Polydispersity index was used as a measure of particle size homogeneity. Data were obtained by averaging 3 measurements at an angle of 90° in cells with 1 cm diameter at 25°C. Samples were diluted approximately 50-fold with distilled water.
To characterize the surface charge of particles, the zeta potential value was obtained by averaging 3 measurements at 25°C. Samples were diluted approximately 200-fold with distilled water.
Nano-CoQ10s were stored in a sealed brown bottle at 25°C for 180 days. Mean particle size, polydispersity index, and zeta potential of the nano-CoQ10s were analyzed at days 1 and 180.
All samples were diluted approximately 100-fold with distilled water. For cryogenic transmission electron microscopy (cryo-TEM), 4
The initial, peak, and terminal temperatures of the reaction and the time necessary for the reaction under the static state were determined by differential scanning calorimetry (DSC) with a DSC Q2000 apparatus (TA Instruments, USA). Each sample (~10 mg) was sealed in an aluminum pan (40
Animal studies were performed in the laboratory animal facility at the Life Science School of Tsinghua University, which obtained Animal Welfare Assurance from the Office of Laboratory Animal Welfare. The local ethics committee approved all animal studies performed. Male Sprague-Dawley rats (
Silicone medical grade tubing (120 mm in length, 0.94 mm O.D. × 0.51 mm I.D., HelixMark) was used to create catheters. For insertion of the catheter in the sinus venosus, the length as measured from the tip of the catheter to the vein was set at 20–30 mm. Rats were anesthetized by intraperitoneal administration of chloral hydrate (100 mg/mL) at a dose of 250 mg/kg body weight. A longitudinal skin incision was made over the area where the right external jugular vein passed dorsally to the pectoralis major muscle. The catheter, filled with 25 units/mL of heparinized physiologic saline, was placed into the right jugular vein and then advanced into the sinus venosus. The catheter was anchored by suturing it to muscle. The free end of the catheter was passed under the skin of the dorsum of the neck just caudal to the ears and attached to the skin. Finally, the catheter was filled with heparinized saline (250 units/mL), and a metal plug was inserted into the free end of the catheter. Rats were placed in individual cages and allowed 24 h to recover from surgery with free access to food and water. At the end of the recovery period, rats were deprived of food overnight.
The next day, the catheter was flushed and filled with heparinized saline (25 units/mL). Thirty-six rats were randomly divided into the following 6 groups (
Blood samples were drawn from the catheter using the following technique. The plunger on a syringe was retracted until a small amount of blood appeared in the needle bulb, and heparin solution was removed from the catheter together with the first sample of blood (30–50
After oral administration of drug, jugular vein blood samples (0.5 mL) were collected from rats and deposited into heparinized microcentrifuge tubes (1.5 mL) at the following time intervals: 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, and 48 h. Blood samples were immediately centrifuged for 10 min at 4000 rpm. Plasma was collected into Eppendorf tubes and immediately stored at −20°C until used for further analyses.
A mixture of plasma (0.1 mL) and an internal standard solution (0.1 mL of a 500 ng/mL methanol solution) was placed in an Eppendorf microtube. Methanol (0.8 mL) was added to precipitate proteins, and the microtube was vortexed for 1 min and centrifuged for 10 min at 12,000 rpm. The supernatant (0.7 mL) was transferred to a vial suitable for liquid chromatography/mass spectrometry (LC/MS).
The quantification of CoQ10, based on a calibration curve of CoQ10 (standard) and CoQ9 (internal standard), was determined by using LC/MS with electrospray ionization system from Agilent. The optimal settings for the MS operated in the positive ion electrospray mode were as follows: gas temperature, 350°C; drying gas flow, 8 L/min; nebulizing gas pressure, 50 psi; sheath gas temperature, 400°C; sheath gas flow, 11 L/min; capillary voltage, 4000 V; nozzle voltage, 500 V. The selected mass-to-charge (
The plasma concentration-time profile was corrected for endogenous levels of CoQ10 as follows. For each animal, the respective endogenous levels of CoQ10 at time 0 h were subtracted from the observed CoQ10 concentrations at each time point. CoQ10 plasma concentrations at different time points for individual rats were analyzed (noncompartmental analysis model) using PKSolver Professional software (China Pharmaceutical University, Nanjing, China). We calculated the area under the plasma concentration-time curve from 0 to 48 h
Nano-CoQ10 formulations were prepared using a high-energy method with a high-pressure homogenizer. Particle size depends primarily on the pressure and the cycle time when a high-energy method is utilized to produce a nanoemulsion [
Physicochemical properties of nano-CoQ10s modified with various surfactants and the stability of nano-CoQ10s during 180 days of storage in sealed brown bottles at 25°C (
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Particle size (nm) | Polydispersity index | Zeta potential (mV) | Particle size (nm) | Polydispersity index | Zeta potential (mV) | |
Nano-CoQ10-TPGS | 66.3 ± 1.5 | 0.197 ± 0.012 | −19.6 ± 1.5 | 72.0 ± 2.0 | 0.201 ± 0.01 | −20.1 ± 1.0 |
Nano-CoQ10-PHCO | 77.3 ± 2.1 | 0.109 ± 0.044 | −12.8 ± 1.4 | 77.7 ± 1.2 | 0.117 ± 0.027 | −13.5 ± 0.7 |
Nano-CoQ10-PSAE | 89.0 ± 3.0 | 0.175 ± 0.014 | −39.1 ± 1.1 | 92.0 ± 1.0 | 0.182 ± 0.007 | −39.5 ± 0.6 |
Nano-CoQ10-SP | 92.7 ± 1.5 | 0.339 ± 0.072 | −37.4 ± 1.0 | 109.3 ± 2.1 | 0.297 ± 0.012 | −37.5 ± 0.9 |
Nano-CoQ10-SL | 88.0 ± 1.0 | 0.527 ± 0.033 | −41.6 ± 1.4 | 102.7 ± 1.5 | 0.453 ± 0.063 | −42.2 ± 0.8 |
The polydispersity index indicates the quality or homogeneity of the dispersion, and a small polydispersity index (less than 0.2) indicates a narrow droplet size distribution [
All nano-CoQ10 formulations had negative surface charges (Table
The stability tests of all nano-CoQ10s were performed at room temperature and evaluated by monitoring the mean particle size, zeta potential, and polydispersity index. In general, nanoemulsions with negative zeta potentials above −30 mV indicate stable formulations [
The advantage of the cryo-TEM methodology is that the liquid dispersion can be frozen and viewed directly in the frozen state; thus, samples can be investigated close to their natural state [
Morphology of nano-CoQ10-PHCO determined by cryo-TEM at 50,000x magnification.
The physical state of CoQ10 in the nano-CoQ10 formulation was investigated because it influences in vitro and in vivo release characteristics and pharmaceutical profiles. DSC curves of CoQ10, surfactant, and nano-CoQ10 are shown in Figure
DSC curves at a heating rate of 5°C/min after 24 h storage at 8°C. (a) Bulk material CoQ10, TPGS, and nano-CoQ10-TPGS; (b) bulk material CoQ10, P-1670, and nano-CoQ10-SP; (c) bulk material CoQ10, SWA-10D, and nano-CoQ10-PSAE; (d) bulk material CoQ10, Cremophor RH40, and nano-CoQ10-PHCO; and (e) bulk material CoQ10, Epikuron 170V, and nano-CoQ10-SL.
The size of nanoparticles plays a key role in their adhesion to and interaction with biological cells. Several possible mechanisms allow particles to pass through the gastrointestinal (and other physiological) barriers. These include paracellular passage that involves particles “kneading” between intestinal epithelial cells due to their extremely small size (<50 nm), endocytotic uptake whereby particles are absorbed by intestinal enterocytes through endocytosis (particle size < 500 nm), and lymphatic uptake whereby particles are adsorbed by M cells in Peyer’s patches (particle size < 5
The pharmacokinetic profiles for orally administered nano-CoQ10s stabilized with different surfactants are shown in Figure
Pharmacokinetics parameters of CoQ10 in rats after a single oral administration of CoQ10 suspension and nano-CoQ10 formulations modified with various surfactants.
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Nano-CoQ10-TPGS | 17.37 ± 2.56 | 6 ± 0 |
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Nano-CoQ10-PHCO | 20.25 ± 1.23 | 7 ± 0 |
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Nano-CoQ10-PSAE | 21.01 ± 2.04 | 6 ± 0 |
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Nano-CoQ10-SP | 20.52 ± 2.78 | 6 ± 0 |
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Nano-CoQ10-SL | 18.52 ± 1.42 | 6 ± 0 |
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CoQ10-Suspension | 18.27 ± 3.71 | 6.33 ± 0.58 | 0.58 ± 0.02 | 11.47 ± 0.77 |
Mean plasma concentration-time profiles of CoQ10 after a single oral administration of nano-CoQ10-TPGS, nano-CoQ10-PHCO, nano-CoQ10-PSAE, nano-CoQ10-SP, nano-CoQ10-SL, or CoQ10-Suspension (60 mg/kg) in Sprague-Dawley rats (
Among the pharmacokinetic parameters assessed,
Lipophilic excipients can have significant and beneficial effects on the absorption and exposure of coadministered lipophilic drugs. After oral administration, the lipophilic drug must first dissolve within the gastrointestinal tract, a physiological and chemical barrier, before partitioning into and then crossing the enterocyte [
CoQ10 was formulated in a lipid-free nano-CoQ10 system in an attempt to increase its solubility and oral bioavailability. Nano-CoQ10 was modified with different surfactants using the hot HPH method. After oral administration in rats, lipid-free nano-CoQ10 significantly improved CoQ10 bioavailability as compared to that following administration of a CoQ10 powder suspension. We determined that surfactants were important for improving CoQ10 bioavailability. Indeed, our lipid-free nano-CoQ10s modified with different surfactants achieved similar or higher levels of CoQ10 bioavailability than that reported for a lipid-based nanoemulsion [
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful for LC/MS support from Yu Tan from the Centre of Biomedical Analysis, Tsinghua University, China. This research was funded by the Suzhou International Science and Technology Cooperation Program (SH201204), Changshu Science and Technology Program (CC201213), and Suzhou Science and Technology Program (ZXG2012034).