Lyotropic Liquid Crystalline Nanoparticles of CoQ10: Implication of

Mar 28, 2014 - ... in the case of GLCQ and PLCQ, respectively, attributed to the formation of the virtual channel pathway as a probable absorption mec...
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Lyotropic Liquid Crystalline Nanoparticles of CoQ10: Implication of Lipase Digestibility on Oral Bioavailability, in Vivo antioxidant activity, and in Vitro−in Vivo Relationships Nitin K. Swarnakar, Kaushik Thanki, and Sanyog Jain* Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab -160062, India S Supporting Information *

ABSTRACT: The present investigation reports implications of the lipase digestibility of lyotropic liquid crystalline nanoparticles (LCNPs) on the oral bioavailability, in vivo antioxidant potential, and in vitro−in vivo relationship (IVIVR) of CoQ10 loaded LCNPs prepared from glyceryl monooleate (GLCQ) and phytantriol (PLCQ). Exhaustive optimization of the process variables was carried out, and optimized lyophilized formulations were found to have particle sizes of 140.45 ± 5.47 nm and 238.42 ± 8.35 nm and a polydispersity index (PDI) of 0.15 ± 0.01 and 0.22 ± 0.03 for GLCQ and PLCQ, respectively. The entrapment efficiency at 10% theoretical loading was found to be >90% in both the cases. The morphological characteristics of the developed formulations were assessed using high resolution transmission electron microscopy and small-angle X-ray scattering analysis, which showed hexagonal (HII) structure. The developed formulations were also found to be stable in simulated gastrointestinal fluids for the stipulated period of time. The in vitro drug release studies revealed a bimodal sustained release drug profile with Higuchi type release kinetics as the best fit release model for both the formulations. The best fit release models were found to be of the Hixson Crowell type in the case of GLCQ when carried out in lipase rich media, suggestive of matrix erosion and subsequent formation of secondary structures, which was further corroborated by carrier degradation studies. Furthermore, 9.1- and 10.67-fold increase in Caco-2 cell uptake was observed in the case of GLCQ and PLCQ, respectively, attributed to the formation of the virtual channel pathway as a probable absorption mechanism. Consequently, 7.09- and 8.67-fold increase in oral bioavailability was observed in the case of GLCQ and PLCQ, respectively. The IVIVR was also established with r2 values in the order of 0.996 and 0.999 for GLCQ and PLCQ, respectively, in contrast to that of 0.484 for free CoQ10. Finally, in vivo prophylactic antioxidant efficacy against the STZtreated rats using various markers such as GSH, LDH, SOD, MDA, glucose level, and body weight showed significantly higher antioxidant activity of CoQ10-LCNPs as compared to that of free CoQ10. In a nutshell, the developed formulation strategy poses great potential in improving the oral bioavailability of difficult-to-deliver drugs such as CoQ10. KEYWORDS: liquid crystalline nanoparticles, coenzyme Q10 (CoQ10), enhanced bioavailability, in vitro−in vivo relationship, in vivo antioxidant activity, Caco-2 cell uptake

1. INTRODUCTION

such imbalances and improve upon cardiovascular health, aging, neurodegenerative diseases, and diabetes conditions.5 Considering the huge potential, CoQ10 has been approved by the US Food and Drug Administration (FDA) for the treatment of various mitochondrial diseases. Although the therapeutic benefits of CoQ10 seems lucrative, oral delivery of CoQ10 is challenging considering its low aqueous solubility (95% in all cases. 2.2.4. Small Angle X-ray Scattering (SAXS). SAXS measurements were performed on a model SAXSess mc2 (Anton Paar GmbH, USA) equipped with a Kratky block-collimation system. The SAXS system comprised a sealed-Cu tube X-ray generator (Philips, PW 1730/10) operating at 40 kV and 50 mA that generates Cu Kα radiation (wavelength 1.54 Å), which was calibrated with a silver behenate standard. The 2D CCD detector featured a 2084 × 2084 array with a 24 × 24 μm2 pixel size at a sample−detector distance of 311 mm and was used to detect signals. Different samples were filled in a capillary sample holder and pre-equilibrated at 25 °C for 30 min and exposed to X-ray beams for 90 min under vacuum. The temperature of the capillary was controlled by using a Peltier system. The scattering files obtained after the analysis of samples were normalized for cosmic ray, detector background, and water background by advanced data interpretation software (PCG). Further, the 2D images of scattering files were processed to the one-dimensional scattering function I(q), where q is the length of the scattering vector, defined by the equation q = (4π/λ) (sin θ/2), with λ being the wavelength and θ the scattering angle. The cubic and hexagonal space groups of liquid crystalline nanoparticles were determined by the relative positions of the 1437

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Table 2. Effect of Solvent on the Preparation of CoQ10-LCNPsa GLCQ

a

PLCQ

type of solvent

size (nm)

PDI

EE (% w/w)

size (nm)

PDI

EE (% w/w)

acetone acetone−ethanol methylene chloride ethyl acetate ethanol

210.14 ± 11.24 196.58 ± 5.21 277.56 ± 14.54 248.33 ± 15.21 875.8 ± 26.97

0.32 ± 0.06 0.26 ± 0.02 0.31 ± 0.04 0.29 ± 0.02 0.38 ± 0.12

69.54 ± 3.21 91.54 ± 2.03 90.54 ± 1.03 91.46 ± 1.63 28.01 ± 4.25

286.54 ± 10.25 282.56 ± 8.54 318.43 ± 17.54 346.67 ± 11.67 986.25 ± 31.25

0.31 ± 0.08 0.23 ± 0.03 0.28 ± 0.05 0.23 ± 0.04 0.47 ± 0.14

72.54 ± 2.41 91.36 ± 2.36 87.65 ± 1.41 88.46 ± 2.43 24.01 ± 4.68

Data are expressed as the mean ± SD (n = 6).

2.3. Caco-2 Cell Culture Experiments. 2.3.1. Cell Culture. Caco-2 cells (American Type Culture Collection) were grown in tissue culture flasks (25 cm2) and maintained under 5% CO2 atmosphere at 37 °C. The growth medium was composed of minimum essential medium Eagle (MEM), 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and amphotericin B (PAA, Austria). The growth medium was changed on every alternate day until confluency was reached. Once confluent, cells were harvested with 0.25% of Trypsin−EDTA solution (Sigma) and either passaged or seeded in cell culture plates for further studies. 2.3.2. Qualitative Cell Uptake. Harvested Caco-2 were seeded in 6 well plates at a cell density of 3,00,000 cells/well and allowed to adhere overnight. The cells were then exposed to free Coumarin 6 (C6), C6 loaded GLCQ, and C6 loaded PLCQ (equivalent to 1 μg/mL C6) and incubated for 4 h. Following the incubation period, the cell culture medium was aspirated, and the cells were washed twice with PBS, fixed with glutaraldehyde (2.5% v/v), and observed under a confocal laser scanning microscope (CLSM) (Olympus FV1000). 2.3.3. Quantitative Cell Uptake. Harvested Caco-2 cells were seeded in a Millicell 24-well cell culture plate equipped with 0.4 μm filter paper (Millipore, USA) at a cell density of 105 cells/well and allowed to form a monolayer over the period of 21 days.35 Transepithelial electrical resistance (TEER) values were monitored for assessing monolayer integrity and confluency. The apical part of cell monolayers were exposed to different concentrations of CoQ10 formulations (at a dose equivalent to 1 and 10 μg/mL CoQ10) and incubated for 0.5, 1, 2, 3, 4, and 6 h. After incubation, monolayers were washed twice with PBS to remove noninternalized drug/CoQ10LCNPs and lysed with 0.1% v/v Triton X-100. Ethanol was implemented as solvent for breaking the matrix of the carrier system. The drug concentration in the cell lysates was estimated by HPLC as described previously.6,36 2.4. In Vivo Pharmacokinetics. Female Sprague−Dawley rats of 220−250 g were supplied by the central animal facility (CAF), NIPER, India. The protocols were duly approved by the Institutional Animal Ethics Committee, National Institute of Pharmaceutical Education & Research (NIPER), India. The animals were acclimatized to standard housing conditions 1 week before experiments. The animals were randomly distributed into three groups each containing 6 animals and received free CoQ10, GLCQ, and PLCQ in a dose equivalent to 15 mg/kg of free CoQ10.6 The blood samples were collected from the retro-orbital plexus under mild anesthesia into heparinized microcentrifuge tubes (containing 30 μL of 1000 U heparin/mL of blood). Plasma was separated by centrifuging the blood samples at 5,000 rcf for 5 min at 15 °C. CoQ10 in the plasma was estimated by employing the validated HPLC method with CoQ9 as the internal standard (Supporting Information, Table S1).6

The pharmacokinetic parameters of plasma concentration− time data were analyzed by a one-compartmental model, using Kinetica software (Thermo scientific, USA). Various required pharmacokinetics parameters like total area under the curve (AUC)0‑∞, time to reach the maximum plasma concentration (Tmax), and peak plasma concentration (Cmax) were determined. 2.5. In Vitro−in Vivo Relationship (IVIVR). The plasma concentration−time profiles for free CoQ10, GLCQ, and PLCQ were implemented to model dependent Wagner Nelson deconvulation procedures for calculating the fraction of drug absorbed as a function of time.37 The equation of the best fit curve for the absorption profile with respect to time was generated using IBM-SPSS Statistics software and utilized for extrapolation of the time which is required for 0.1× fraction of the drug to be absorbed. Similar sets of statistical treatments were also implemented in the in vitro release profile of CoQ10 obtained from free CoQ10, GLCQ, GLCQ (in lipase rich media), PLCQ, and PLCQ (in lipase rich media). Levy plots were further constructed to identify the IVIVR in all of the formulations on the basis of the values of correlation coefficients.38 2.6. In Vivo Antioxidant Activity of CoQ10 Formulations. The in vivo antioxidant potential of CoQ10 formulations was evaluated in terms of their ability to counterfeit the oxidative stress produced in a streptozotocin (STZ) challenge SD rat model as per the earlier reported protocol.39 Briefly, Sprague−Dawley rats were randomly divided into five groups each containing six animals. Group 1 was considered as the negative control and received only the vehicle (PBS, pH 7.4). Groups 2, 3, 4, and 5 received prophylactic treatment of PBS (pH 7.4), free CoQ10, GLCQ, and PLCQ (p.o. 15 mg/kg equivalent to CoQ10). On the next day, the animals of groupd 2−5 were subjected to STZ challenge at 50 mg/kg, i.p. prepared in ice cold 10 mmol citrate buffer at pH 4.4. The animals were humanely sacrificed after 72 h of STZ dosing. The pancreases from the animals were excised, washed, and processed for the estimation of SOD, GSH, LDH, and MDA.36 The blood samples were also collected by cardiac puncture and processed for the estimation of TNF-α and nitric oxide (NO) using commercially available kits following the manufacturer’s instructions (eBioscience Inc. San Diego, CA).36 In a separate set of experiments, the plasma blood glucose levels and body weight were also noted for STZ challenged animals for 3 weeks.40 2.7. Statistical Analysis. All in vitro and in vivo data are expressed as the mean ± standard deviation (SD) and mean ± standard error of the mean (SEM), respectively. Statistical analysis was performed with Sigma Stat (version 2.03) using one-way ANOVA followed by a Tukey−Kramer multiple comparison test. p < 0.05 was considered as a statistically significant difference. 1438

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Table 3. Effect of the Type of Surfactant on Particle Size, PDI, Zeta Potential, Phase Behavior, and Entrapment Efficiency of CoQ10-LCNPsa GLCQ

a

PLCQ

type of surfactant

size (nm)

PDI

EE (% w/w)

size (nm)

PDI

EE (% w/w)

Pluronic F127 Pluronic F-108 Pluronic F-68

196.58 ± 5.21 193.25 ± 7.32 190.65 ± 4.89

0.26 ± 0.02 0.24 ± 0.02 0.27 ± 0.13

91.54 ± 2.03 92.34 ± 1.67 98.88 ± 3.38

282.56 ± 8.54 294.25 ± 7.32 256.98 ± 5.76

0.23 ± 0.03 0.22 ± 0.09 0.20 ± 0.04

91.36 ± 2.36 92.61 ± 2.19 98.56 ± 1.86

Data are expressed as the mean ± SD (n = 6).

3. RESULTS 3.1. Optimization of Formulation Components and Process Variables. CoQ10 exhibits very low solubility in most of the organic solvents thereby limiting the choice of suitable solvents. Among the available solvents, maximum solubility was observed in the case of acetone as compared to that of methylene chloride, ethyl acetate, and ethanol. Although the particle size noted in the case of acetone was in the desired range, the entrapment efficiency was compromised to a greater extent (Table 2). In contrast, water immiscible solvents (methylene chloride and ethyl acetate) resulted in a significant (p < 0.05) appreciation in entrapment efficiency but produced larger sized particles and higher PDI along with some visible aggregates. Marked improvement in particle size, PDI, and entrapment efficiency was observed upon employing an acetone−alcohol mixture as solvent as compared to the plain solvents and hence was used for further studies. Notably, the particle size in the case of GLCQ was significantly (p < 0.05) lower than that of PLCQ, while no statistical significance (p > 0.05) was observed in the case of PDI and entrapment efficiency. The type of surfactant employed during the formulation had significant (p < 0.05) impact on the entrapment efficiency of the CoQ10 for both the lipids (Table 3). However, no statistically significant (p > 0.05) changes in particle size and PDI were observed. Maximum entrapment efficiency of CoQ10 was observed in the case of Pluronic F-68 for both the lipids; however, the requisite concentration for the desired quality attributes of particle size, PDI, and entrapment efficiency varied remarkably. As evident from Table 4, the optimum concentration of Pluronic F-68 was found to be 0.20% w/v and 0.6% w/v for GLCQ and PLCQ, respectively.

Table 5 reflects the effect of drug loading on the quality attributes of GLCQ and PLCQ. Significant decrease (p < 0.05) in the particle size and PDI was observed with increase in drug loading in both the cases until 10% w/w without any compromise in entrapment efficiency. Beyond this concentration, both the lipids performed differently with drastic reduction in entrapment efficiency in the case of GMO in contrast to complete precipitation in the case of Phytantriol. Interestingly, the conversion of cubic to hexagonal structure was also observed in the case of CoQ10-LCNPs upon increasing the theoretical drug loading measured as a function of increase in anisotropy (Table 5). Hence, 10.0% w/w drug loading was implemented for further studies. 3.2. Lyophilization of CoQ10-LCNPs. Cryoprotectant screening suggested the potential of mannitol in retaining the original quality attributes for both the optimized formulations (Supporting Information, Figure S1). Further, 5% w/v concentration of mannitol resulted in the minimum Sf/Si ratio (ratio of particle size after and before freeze-drying), i.e., 1.16 and 1.13 for GLCQ and PLCQ, respectively, with no statistically significant changes (p > 0.05) in entrapment efficiency upon lyophilization (Table 6). Furthermore, lyophilization resulted in an intact fluffy cake which reconstituted in 80%). 3.6. In Vitro Release. A sustained biphasic release profile composed of a relatively rapid release in initial 12 h, i.e., 29.43 ± 2.3% in the case of GLCQ and 19.24 ± 2.11% in the case of PLCQ, followed by a delayed release until 120 h was observed (Figure 3). Free CoQ10 was also utilized as the control which revealed >95% release within 2 h from the dialysis membrane when presolubilized in 5% v/v cremophore EL. However, considering the poor physicochemical properties of CoQ10, 1440

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Figure 2. Small angle X-ray analysis of CoQ10-LCNPs shows (A) diffraction peaks with their Miller indices and (B) validation of the assigned Miller indices.

Table 7. Stability Studies of CoQ10-LCNPs Formulations in Various GIT Mediaa final

initial

a

conc. (% w/v)

size (nm)

PDI

EE (%w/w)

SGF (2 h) SIF (6 h) SGF (2 h) + SIF (4 h)

140.45 ± 5.47

0.15 ± 0.01

GLCQ 92.54 ± 2.37

SGF (2 h) SIF (6 h) SGF (2 h) + SIF (4 h)

238.42 ± 8.35

0.22 ± 0.03

PLCQ 96.37 ± 2.67

size

PDI

EE (% w/w)

139.56 ± 6.19 132.23 ± 5.32 131.34 ± 5.86

0.19 ± 0.02 0.27 ± 0.02 0.28 ± 0.05

87.14 ± 2.68 84.74 ± 2.42 80.34 ± 2.32

252.71 ± 6.15 246.23 ± 7.81 245.56 ± 6.54

0.23 ± 0.03 0.18 ± 0.03 0.19 ± 0.06

95.34 ± 2.32 93.62 ± 2.71 93.12 ± 2.32

Data are expressed as the mean ± SD (n = 6).

viability was observed in all formulations suggesting the interaction of LNCPs with cells. Therefore, the effect of various concentrations of LCNPs and their incubation time on the cells were evaluated, which revealed concentration dependent alteration in the cell morphology (Figure 5B). Exposure of cells with lipid concentration (below the 80 μg/ mL) led to alteration in the cell morphology which was found to be reversible in nature and hence demonstrated >90% cell viability. Furthermore, the magnitude of the interaction was found to be higher upon exposure of cells with increasing concentrations of lipid (80−100 μg/mL). At this threshold concentration, visible changes in the cell morphology were observed leading to reduced cell viability (∼40% and ∼36% in the case of blank Phytantriol- and GMO-based LCNPs at 100 μg/mL of lipid) (Figure 5B, C, and F). 3.9. In Vivo Pharmacokinetics. The plasma concentration profiles of CoQ10 after single oral administration of the CoQ10-LCNPs and free CoQ10 suspension at 15 mg/kg are shown in Figure 6, and the obtained parameters are

Coumarin-6 (C-6), C-6 loaded GLCQ, and C-6 loaded PLCQ. The images reveal higher intracellular uptake of CoQ10-LCNPs as compared to that of free C-6. Further, concentration and time dependent cellular uptake of CoQ10 by Caco-2 cells was observed in the case of CoQ10 formulations. However, the steady state intracellular concentration of CoQ10 was varied depending on CoQ10 formulations (Figure 5A). Significantly higher (p < 0.001) steady state concentration of CoQ10 was observed in the case of CoQ10-LCNPs (548.89 ± 16.35 and 468.31 ± 9.03 ng/mL in the case of PLCQ and GLCQ, respectively, at 4 h of incubation) as compared to free that of CoQ10 (51.49 ± 2.9 ng/mL at 6 h) at 10.0 μg/mL. The level of CoQ10 was 10.67- and 9.1-fold higher in the case of PLCQ and GLCQ, respectively, as compared to that of free CoQ10 (Figure 5E). Furthermore, cell viability was also found to be >90% in all of the cases indicative of the absence of any toxicity to Caco-2 cells at the tested concentration (10.0 μg/mL). The studies at higher concentration of CoQ10-LCNPs and blank LNCPs were also tried, but marked reduction in the cell 1441

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compared to that of free CoQ10, which could be detected only until 36 h (Figure 6). The mean AUC0−∞ values for GLCQ and PLCQ were 22751.84 ± 604.01 ng/mL·h and 27643.14 ± 936.7 ng/mL·h, respectively, revealing 7.09- and 8.67-fold higher bioavailability as compared to that of free CoQ10 (Table 9). The typical profile of GLCQ demonstrated higher performance up to Cmax as compared to that of PLCQ; however, rapid decline in the plasma concentration of CoQ10 was observed leading to a mean AUC value less than that of PLCQ. 3.10. IVIVR. The IVIVR of the formulations established using Levy’s plot suggested high correlation coefficients for GLCQ (r2 = 0.957) and PLCQ (r2 = 0.999) in contrast to that of free CoQ10 (r2 = 0.4842) (Figure 7). The value of the correlation coefficient increased in the case of GLCQ (r2 = 996) when data of the in vitro release profile in lipase rich media were fitted in to calculations, whereas no such effect was observed in the case of PLCQ (Figure 4). 3.11. In Vivo Antioxidant Activity of CoQ10 Formulations. Significantly (p < 0.001) higher levels of LDH and MDA and lower levels of GSH and SOD in pancreatic tissue as compared to that of the negative control evidenced the oxidative stress following STZ treatment (positive control) (Figure 8A−D). The levels of secretary biomarkers, i.e., TNF-α and NO also increased drastically in plasma as compared to that of the control (Figure 8E−F). The levels of these biochemical markers were insignificantly (p > 0.05) changed in the cases of free CoQ10-pretreated animals as compared to that of the positive control. Interestingly, the levels of biochemical markers in the cases of both GLCQ- and PLCQ-pretreated animals were significantly (p < 0.001) lowered as compared to that of both free CoQ10-pretreated rats and shifted toward the normal values. Of note, the in vivo antioxidant potential of PLCQ was found to be significantly higher (p < 0.05) as compared to that of GLCQ. The alterations in the levels of various biochemical parameters for assessing the oxidative stress were in tandem with the gradual decrease in body weight and the plasma glucose levels, which were found to be >250 mg/dL in the case of STZ challenged rats as compared to those of the negative control throughout the duration of experiment, i.e., up to 3

Figure 3. In vitro release profile of free CoQ10 suspension, free CoQ10 (presolubilized), GLCQ, GLCQ (in lipase rich media), PLCQ, and PLCQ (in lipase rich media). Each data point represents the mean ± SD (n = 6).

Table 8. Drug Release Parameter (Correlation Coefficient) CoQ10-LCNPs after Fitting in Various Release Models

a

particle size of formulation

zero order

first order

Higuchi

HixsonCrowell root

GLCQa

0.754

0.917

0.956

0.933

GLCQb

0.748

0.899

0.943

0.985

PLCQa

0.803

0.922

0.964

0.936

PLCQb

0.814

0.931

0.959

0.941

Korsmeyer Peppas 0.929, n = 0.541 0.975, n = 0.484 0.615, n = 0.614 0.618, n = 0.627

In biorelevant media. bIn lipase rich media.

summarized in Table 9. Significantly higher (p < 0.001) Cmax in the order of 6.5- and 5.0-fold was observed in the case of GLCQ and PLCQ as compared to that of free CoQ10, respectively. Moreover, sustained plasma profile of the drug up to 72 was observed in the case of GLCQ and PLCQ as

Figure 4. In vitro digestion studies of (A) GLCQ and (B) PLCQ in the presence of lipase. 1442

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Figure 5. Uptake of the formulation in Caco-2 cells. (A−D) CLSM images of the cells after 4 h of incubation with (i) free Coumarin-6, (ii) Coumarin-6-GLCQ, and (iii) Coumarin-6-PLCQ. The representative images (iv), (v), and (vi) are the overlays of the corresponding fluorescent and DIC image; (B,C) differential interference contrast (DIC) images of the cells at 20× showing alteration in cell morphology after 4 h of incubation with blank PLCNPs and GLCNPs, respectively, at a concentration of (i) 50 μg/mL, (ii) 100 μg/mL, and (iii) 200 μg/mL; while (D) depicts the image of control cells. The inset image shows an enlarged view of representative cells. (E) Concentration and time dependent quantitative uptake of the CoQ10-formulation in Caco-2 cells; (F) concentration and time dependent cytotoxicity of GLCNPs and PLCNPs in Caco-2 cells. ***p = 0.001, **p = 0.01; (a) vs free CoQ10 (50 μg/nL), (b) vs GLCQ (1 μg/mL), (c) vs PLCQ (1 μg/mL), and (d) vs GLCQ (10 μg/mL). Each data point is the mean ± SD (n = 6). (B,D) Adapted with permission from ref 30. Copyright 2013 American Association of Pharmaceutical Scientists.

weeks (Figure 9). Interestingly, increased plasma glucose levels up to 1 week followed by a gradual decrease toward the normal values was observed in the case of GLCQ- and PLCQpretreated animals (Figure 9A). These results were also well corroborated with the observed body weight of animals (Figure 9B). As anticipated, the normalization in the levels of plasma glucose and body weight was significantly higher (p < 0.05) in the case of PLCQ as compared to that of GLCQ.

4. DISCUSSION The solvent diffusion−evaporation method was utilized for the preparation of CoQ10-LCNPs with solvents acting as thinning agents thereby decreasing the viscosity of the organic phase (lipid and drug) and increasing the dispersibility of the organic phase upon addition into the stabilizer solution. Considering the solubility of drug and lipid, acetone, methylene chloride, ethyl acetate, and ethanol were screened as solvents. Among the tested solvents, a gradual increase in the entrapment efficiency was observed as a function of decrease in water miscibility; however, the proportionate increase in the particle size was also

Figure 6. Plasma concentration−time profiles of free CoQ10, GLCQ, and PLCQ after oral administration to SD rats at 15 mg/kg dose. Each data point is expressed as the mean ± SEM (n = 6).

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Table 9. Pharmacokinetic Parameters after Oral Administration of Free CoQ10, GLCQ, and PLCQa

a

parameters

free CoQ10

GLCQ

PLCQ

Cmax (ng/mL) Tmax (h) AUC0‑t (ng/mL·h) AUCtot (ng/mL·h)

140.40 3 3293.19 3431

916.6 ± 57.33 4 19144.1 ± 489.22 22751.84 ± 604.01

692.4 ± 77.73 8 20261.65 ± 895.3 27643.14 ± 936.7

Values are expressed as the mean ± SEM (n = 6).

Figure 7. IVIVR among various CoQ10 formulations.

assembly.44 Diffusion of the solvent leads to the nucleation of the lipid matrix, which needs to be stabilized by a suitable surfactant. In present study, high molecular weight triblock copolymers (Pluronic series) were used owing to their tendencies to preferentially adsorb on LCNPs surface, which prevents unwanted changes in the liquid crystalline structure.45 Among the tested surfactants, maximum entrapment efficiency was observed in the case of Pluronic F-68 owing to the minimum solubility of the drug (56.86 μg/mL) in comparison to that of Pluronic F-108 (89.56 μg/mL) and Pluronic F-127 (98.56 μg/mL). Hence, Pluronic F-68 minimized the leaching of the drug from the matrix of the lipid and led to highest

evident concomitantly (Table 2). The probable reason for this observation could be rapid diffusion of water miscible solvents along with drug into the surfactant solution. In principle, slow solvent diffusion along with rapid formation of lipid assembly is required to achieve maximum entrapment efficiency. Therefore, the maximum permissible amount of ethanol (without any drug precipitation in solvent) was supplemented with acetone, and notably, this strategy resulted in superior entrapment efficiency without any compromise in particle size, which could be attributed to the relatively higher viscosity of solvent as compared to that of acetone alone (Table 2). In addition, the solvent polarity also contributed to the formation of lipid 1444

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Figure 8. Levels of various biochemical parameters in pancreatic tissue (A) LDH, (B) MDA, (C) GSH, and (D) % SOD; in plasma (E) nitric oxide (NO) production; and (F) TNF-α levels, after the third day of prophylactically treated STZ challenged rats. ***p < 0.001, *p < 0.05; (a) vs control, (b) vs STZ treated rats, (c) vs free CoQ10 + STZ treated rats, and (d) vs GLCQ + STZ treated rats. Each data point is the mean ± SD (n = 6).

Figure 9. Changes in plasma glucose levels and total body weight in prophylactic treated-STZ challenged rats. ***p < 0.001, **p 10% w/w beyond which a significant (p < 0.05) increase in the particle size and PDI were noted (Table 5). Of note, the changes in the phase behavior are suggestive of the incorporation of lipophilic CoQ10 into the liquid crystalline phase. The observations were further confirmed by SAXS studies, which could be attributed to the interaction of lipophilic drug with a hydrophobic tail of lipid leading to an increased value of the critical packaging parameter (Table 5). Hence, this altered molecular packing of the lipids results in the

entrapment efficiency (Table 3). A similar type of relationship between the drug solubility in the surfactant and entrapment efficiency of the drug in the nanoformulation was also reported earlier.30,46 Further, the stabilization effects were evident by improvement in the PDI of the formulation with increase in the concentration of Pluronic F-68 (Table 4). Subsequently, optimization of drug loading in the LCNPs was also performed to maximize the drug loading within the formulation. The results revealed a saturation of the drug space within LCNPs 1445

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TEM examination of GLCQ and PLCQ was carried out to investigate their structural stability in biorelevant media supplemented with lipase, which revealed time dependent alterations in the structures of GLCQ as a function of time (Figure 4). No visible changes were observed in SGF supplemented with 50 IU/mL lipase up to 2 h; however, the presence of small sized (∼70 to 125 nm) secondary structures were observed until 8 h in the presence of SIF (supplemented with 1000 IU/mL lipase) suggesting partial digestion of GLCQ. The partial retention of the structure could be explained on the basis of a complex arrangement of lipid in the form of a matrix and the presence of bulky surfactant on the surface restricting the extent of lipase action on GMO which otherwise is rapidly degraded.23 Of note, no such alterations in the PLCQ were observed (Figure 4). Notably, this transformation is often attributed to GI instability and accounted for the negative performance of the GMO based formulations, as observed in the case of cinnarazine, where practically no appreciation in bioavailability enhancement was noted.24 The stability of lipid in the presence of lipase has been regarded as the principal reason for this observation to date.17,18 However, contradictory results were observed in our case, and no precipitation of CoQ10 was observed even in the case of GMO based formulations in GIT fluids. Upon further indepth investigation and one to one comparison, we identified the importance of yet another aspect, i.e., the solubility of the drug in GMO as a critical variable in predicting the performance of the LCNPs. On the basis of the fact that GMO solubility of CoQ10 is ∼5fold higher than that of cinnarizine,17 we hypothesize that it is the solubility which is probably responsible for the retention of the drug upon digestion of GMO matrices by lipases and conversion into suitable secondary structures. The said hypothesis was further supported by in vitro drug release studies. Significant increase in the solubilization potential along with faster release of CoQ10 was observed in the case of GLCQ and PLCQ in contrast to that of free the CoQ10 suspension. Interestingly, when presolubilized CoQ10 in 5% v/v Cremophore EL was employed, complete release was observed within 2 h suggestive of poor wetting and solubility limited dissolution of CoQ10. Of note, presolubilized CoQ10 tends to precipitate in simulated gastrointestinal fluids at extreme dilutions and hence was not considered for further studies. Furthermore, the release was relatively higher in the case of GLCQ when exposed to the lipase enzyme, indicative of the interaction of CoQ10 with GMO matrices. The release kinetics revealed the Hixson−Crowell model as the best fit for GLCQ in the presence of lipase suggesting erosion of the lipid matrix with time (Figures 3 and 4, and Table 8). In contrast, both GLCQ and PLCQ preferentially followed the Higuchi model indicative of the matrix dependent diffusion of the drug, which could be attributed to a systematic arrangement of the lipid in the matrix of LCNPs that provides tortuous diffusion pathways for the drug (Figures 1 and 2). The oral delivery potential of CoQ10-LCNPs was evaluated in a in vitro Caco-2 cell culture model which was widely accepted as a surrogate for assessing the uptake and transepithelial transport of various bioactives and nanoparticles.30,52 The results revealed a significantly higher uptake of CoQ10-LCNPs as compared to free CoQ10 at all tested concentrations (Figure 5A and E), suggesting the involvement of additional uptake mechanisms in the case of CoQ10-LCNPs. Furthermore, CLSM studies also revealed the morphological changes in the cell membrane (altered membrane integrity)

formation of hexagonal shaped LCNPs instead of cubic shape.15,16 Further, the observed changes in the LCNPs were confirmed by SAXS analysis, which revealed progressive changes in the spectra (data not shown). Subsequently, the effect of sonication time on the quality attributes of the formulation was also studied (Table 10). The optimized formulation was further lyophilized using a stepwise freezedrying cycle.28 Evaluation of the lyophilized product demonstrated that the all-original quality attributes of formulations such as particle size, PDI, and entrapment efficiency were maintained when 5% w/v mannitol was implemented as cryoprotectant. Furthermore, the lyophilized formulation demonstrated 6 months of accelerated stability as per ICH guidelines (Supporting Information, Table S2). Morphological examination of the CoQ10-LCNPs by HRTEM revealed their hexagonal geometry with evidence of water channels within the structure (Figure 1). Notably, marginal discrepancies in the dimensions were observed as compared to that from the zeta sizer results (140.45 ± 5.47 nm in the case of GLCQ and 238.42 ± 8.35 nm in the case of PLCQ), which could be attributed to the typical operating principles of used techniques. The sample is dried and is visualized under vacuum in the case of TEM leading to a slight decrease in the observed size, whereas in contrast, hydrodynamic volume of particles is measured in the case of the zeta sizer.47,48 The crystallinity in the CoQ10-LCNPs was further confirmed by SAXS analysis.15,36,49 The spectral pattern of CoQ10-LCNPs depicted three diffraction peaks with a relative ratio of 1:31/2:2 suggestive of HII type hexagonal geometry in the system (Figure 2A). Furthermore, assignment of crystallographic space groups, i.e., Miller indices as (h, k, and l) = (1, 0, 0), (1, 1, 1), and (2, 0, 0) for CoQ10-LCNPs was considered to be valid (Figure 2B) because points of the graph demonstrated a linear trend line (r2 values >0.99), which intercepts at origin (0,0). The slightly lesser values of the lattice constant (∼0.84 and ∼0.5 Å in the case of GLCQ and PLCQ, respectively) in comparison to their respective blank LCNPs reflected dehydration (or shrinking of the water channels) in the system as reported previously.15,50 This dehydration also suggested that the incorporation of lipophilic CoQ10 increases the hydrophobic volume of polar lipids (GMO or Phytantriol) which could also be correlated with increased critical packaging parameters of the system and conversion of geometry of the system from cubic to hexagonal shape (Table 5). Furthermore, the observed structural information (cubic and hexagonal geometries) of the system also well corroborates with the results of polarized microscopy which showed isotropic (dark background) and anisotropic structures (fan-like structure and angular texture) in the case of liquid crystalline phase (Supporting Information, Figure S2). In vitro stability studies in simulated gastrointestinal fluids revealed the robustness of formulation in GIT fluids with insignificant change (p > 0.05) in quality attributes upon incubation for a stipulated time period with respective SGF/SIF (Table 7). The entrapment efficiency of CoQ10-LCNPs was maintained (>80%) due to the characteristic features of LCNPs, which include sustained release characteristics and surfactant assisted steric stabilization of the nanoparticles.24,51 The marginal reduction in entrapment efficiency in the case of GLCQ in comparison with that of PLCQ could be attributed to the labile nature of GMO in the presence of biorelevant media. In addition, a slight decrease in the particle size with increase in PDI also revealed transformation into secondary structures and is in line with the previously reported literature.23 Hence, HR1446

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pancreas usually contains very low levels of antioxidant enzymes as compared to other types of cells and hence is more susceptible to reactive oxygen species.57,58 Mechanistically, STZ induces severe reactive oxygen species leading to excessive DNA damage and depletion of vital bioactives including CoQ10.59 The deficiency of CoQ10 in the pancreas could impair bioenergetics such as reduction in biosynthesis of ATP and insulin leading to decreased functionality of beta cells. Furthermore, the hyperglycemia dependent increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to the generation of excessive superoxide in the cells. 60 Therefore, significant decrease in the level of antioxidants (GSH and SOD) and significantly increased levels of oxidized toxic product (LDH and MDA) and secretary mediators (TNF-α and NO) are noted in the case of STZ challenged rats (Figure 8). As a consequence, plasma glucose levels often shoot >250 mg/dL with concomitant decline in the body weight (Figure 9). The consistently increased plasma glucose levels are indicative of the complete degranulation of beta cells and development of permanently diabetes.39 Further, no appreciation in the glucose levels and body weight was observed in the prophylactically treated free CoQ10 group, which could be correlated with its poor bioavailability (Figure 6). In contrast, prophylactic treatment of developed formulations showed decreased maximal free radical burst in the early periods of STZ administration, and the magnitude of efficacy was found to be significantly higher than that of the positive control Figure 8). Thus, significantly higher protective antioxidant effects (normalized biochemical parameters and lowered glucose level) as compared to the pre-free CoQ10 treated group (p < 0.001). Of note, the magnitude of the normalization plasma glucose levels was significantly higher (p < 0.05) in the case of PLCQ than that of GLCQ. Furthermore, the % diabetic population in the case of PLCQ was ∼33% (2 out of 6) in contrast to that of ∼66% (4 out of 6) in the case of GLCQ and 100% (6 out of 6) in the case of positive control. However, the % diabetic population in both cases of GLCQ and PLCQ was nil within 3 weeks. Interestingly, the increased early glucose levels were further decreased toward the normal level after 3 weeks, which could also be correlated with antioxidant properties of CoQ10-LCNPs that might have protected the viability of beta cells above the certain threshold viability. This protection of beta cells could also maintain some functionality of beta cells which may be further rejuvenated to normal level with time as reported earlier.39 Cumulatively, the higher stability of the carrier and sustained plasma profile and bioavailability of CoQ10 in PLCQ are suggestive of its superior delivery potential as compared to that of GLCQ.

during the uptake phase of LCNPs, which were referred to as “virtual paths” (Figure 5 B, C, and D). More interestingly, the virtual paths were reversed with time, and normal membrane physiology was attained within 12 h (data not shown). Thus, based on these observations a mechanism of uptake via the formation of some reversible “virtual pathways” in the cell membrane was responsible for a significantly higher uptake of CoQ10 by LCNPs. The said observation could be correlated with membrane modifying (fluidizing or fusogenic) properties of LCNPs as reported earlier15,53,54 along with other uptake mechanisms such as clathrin and caveolae/lipid raft-mediated endocytosis53 and transportation across the cells by long chain fatty acid transporters. 55 Most interestingly, all tested concentrations of CoQ10-LCNPs or free CoQ10 did not cause any significant toxic effect on the Caco-2 cells, indicating the safety of the lipid/drug loaded formulation. Moreover, both lipids are also categorized under a category that is generally recognized as safe (GRAS) for oral administration.25,26 The enhanced Caco-2 uptake of CoQ10-LCNPs was well corroborated with in vivo pharmacokinetics. Significantly higher (p < 0.05) Cmax and sustained plasma levels of CoQ10 up to 72 h were achieved in the case of CoQ10-LCNPs as compared to those of free CoQ10. Furthermore, the relative oral bioavailability of CoQ10 was found to be 7.09-fold and 8.67fold higher in the case of GLCQ and PLCQ, respectively, as compared to that of free CoQ10 (Table 9). Although, rapid absorption of GLCQ was observed in the order of 132% higher Cmax as compared to that of PLCQ, a slight decrease in the relative oral bioavailability was observed, which could be attributed to the partial digestibility of GLCQ in the presence of lipase and plasma esterases (Figure 4). Of note, the presence of GLCQ and their secondary structures which are bioadhesive in nature increases the opportunities of close contact of the drug loaded LNCPs with the endothelial cell membrane and could overcome the “unstirred water layer” barrier56 and hence demonstrated a high delivery potential in the initial 4 h. However, afterward most of the structural properties of the carrier were lost owing to the higher digestion capability of pancreatic lipase which could also be correlated with the abrupt decline of the plasma level of CoQ10 after 6 h. Thus, based on this observation we attempted the correlation of the in vitro release profile with the in vivo performance of carrier. The Levy’s plot of free CoQ10 showed a poor correlation coefficient; in contrast, the incorporation of a drug in LCNPs led to high IVIVR (r2 >0.9). The possible reason for this value of IVIVR could also be correlated with unique properties of LCNPs viz. lipid-mixing, membrane-fusing, and formation of “virtual pathway” in the membrane which might also helped in enhancing the bioavailability of the drug (Figure 5B and C). Further, the relationship was fitted more accurately (r2 = 0.9961) in the case of GLCQ if data of the release study in the presence of lipase rich media was utilized, while PLCQ demonstrated better IVIVIR (r2 > 0.99) in all cases owing to the chemical stability in the presence of lipase (Figures 4 and 7). Therefore, implementation of a later strategy would be desired in the case of digestible lipid for better IVIVR prediction. Upon correlating the effect of lipase digestibility on the in vivo pharmacokinetic profiles of the developed formulations, the obvious concern on the in vivo antioxidant potential was also addressed. The STZ induced diabetic model is well reported for type I diabetics and is closely associated with oxidative stress by STZ on the pancreatic beta cells. The

5. CONCLUSIONS The present study reports a one-to-one comparison of digestible and digestion-resistant lipids and its influence on the overall in vitro and in vivo performance of developed LCNPs. By virtue of exhaustive optimization, in vitro and in vivo evaluations, it could be concluded that the solubility of the drug in the lipid matrices apart from the digestibility also plays an important role while designing and developing LCNPs. Furthermore, the lyophilization of LCNPs and established IVIVR could suitably be applied to assist the rapid development of LCNPs from industrial perspectives. Mechanistic understanding on the influence of secondary structures in the bioavailability enhancement could be further explored to have deeper insights on formulated LCNPs. Furthermore, ligand 1447

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(10) Terao, K.; Nakata, D.; Fukumi, H.; Schmid, G.; Arima, H.; Hirayama, F.; Uekama, K. Enhancement of oral bioavailability of coenzyme Q10 by complexation with [gamma]-cyclodextrin in healthy adults. Nutr. Res. (N.Y.) 2006, 26 (10), 503−508. (11) Gokce, E. H.; Ozyazici, M.; Souto, E. B. Nanoparticulate strategies for effective delivery of poorly soluble therapeutics. Ther. Delivery 2010, 1 (1), 149−167. (12) Bhandari, K. H.; Newa, M.; Kim, J. A.; Yoo, B. K.; Woo, J. S.; Lyoo, W. S.; Lim, H. T.; Choi, H. G.; Yong, C. S. Preparation, characterization and evaluation of coenzyme Q10 binary solid dispersions for enhanced solubility and dissolution. Biol. Pharm. Bull. 2007, 30 (6), 1171−1176. (13) Onoue, S.; Uchida, A.; Kuriyama, K.; Nakamura, T.; Seto, Y.; Kato, M.; Hatanaka, J.; Tanaka, T.; Miyoshi, H.; Yamada, S. Novel solid self-emulsifying drug delivery system of coenzyme Q(10) with improved photochemical and pharmacokinetic behaviors. Eur. J. Pharm. Sci. 2012, 46 (5), 492−499. (14) Thanki, K.; Gangwal, R. P.; Sangamwar, A. T.; Jain, S. Oral delivery of anticancer drugs: challenges and opportunities. J. Controlled Release 2013, 170 (1), 15−40. (15) Swarnakar, N. K.; Jain, V.; Dubey, V.; Mishra, D.; Jain, N. K. Enhanced oromucosal delivery of progesterone via hexosomes. Pharm. Res. 2007, 24 (12), 2223−2230. (16) Jain, V.; Swarnakar, N. K.; Mishra, P. R.; Verma, A.; Kaul, A.; Mishra, A. K.; Jain, N. K. Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy. Biomaterials 2012, 33 (29), 7206−7220. (17) Nguyen, T. H.; Hanley, T.; Porter, C. J.; Larson, I.; Boyd, B. J. Phytantriol and glyceryl monooleate cubic liquid crystalline phases as sustained-release oral drug delivery systems for poorly water soluble drugs I. Phase behaviour in physiologically-relevant media. J. Pharm. Pharmacol. 2010, 62 (7), 844−855. (18) Porter, C. J.; Trevaskis, N. L.; Charman, W. N. Lipids and lipidbased formulations: optimizing the oral delivery of lipophilic drugs. Nat. Rev. Drug Discovery 2007, 6 (3), 231−248. (19) Lai, J.; Lu, Y.; Yin, Z.; Hu, F.; Wu, W. Pharmacokinetics and enhanced oral bioavailability in beagle dogs of cyclosporine A encapsulated in glyceryl monooleate/poloxamer 407 cubic nanoparticles. Int. J. Nanomedicine 2010, 5, 13−23. (20) Lian, R.; Lu, Y.; Qi, J.; Tan, Y.; Niu, M.; Guan, P.; Hu, F.; Wu, W. Silymarin glyceryl monooleate/poloxamer 407 liquid crystalline matrices: physical characterization and enhanced oral bioavailability. AAPS PharmSciTech 2011, 12 (4), 1234−1240. (21) Lai, J.; Chen, J.; Lu, Y.; Sun, J.; Hu, F.; Yin, Z.; Wu, W. Glyceryl monooleate/poloxamer 407 cubic nanoparticles as oral drug delivery systems: I. In vitro evaluation and enhanced oral bioavailability of the poorly water-soluble drug simvastatin. AAPS PharmSciTech 2009, 10 (3), 960−966. (22) Tamayo-Esquivel, D.; Ganem-Quintanar, A.; Martinez, A. L.; Navarrete-Rodriguez, M.; Rodriguez-Romo, S.; Quintanar-Guerrero, D. Evaluation of the enhanced oral effect of omapatrilat-monolein nanoparticles prepared by the emulsification-diffusion method. J. Nanosci. Nanotechnol. 2006, 6 (9−10), 3134−3138. (23) Nguyen, T. H.; Hanley, T.; Porter, C. J.; Larson, I.; Boyd, B. J. Phytantriol and glyceryl monooleate cubic liquid crystalline phases as sustained-release oral drug delivery systems for poorly water-soluble drugs II. In-vivo evaluation. J. Pharm. Pharmacol. 2010, 62 (7), 856− 865. (24) Nguyen, T. H.; Hanley, T.; Porter, C. J.; Boyd, B. J. Nanostructured liquid crystalline particles provide long duration sustained-release effect for a poorly water soluble drug after oral administration. J. Controlled Release 2011, 153 (2), 180−186. (25) Final report on the safety assessment of phytantriol. Int. J. Toxicol. 2007, 26 (1), 107−114. (26) US Department of Agriculture. Glycerol Monooleate Processing. http://www.ams.usda.gov/AMSv1.0/getfile?dDocName= STELPRDC5057603 (accessed on 09 Dec, 2013).

anchored and surfactant assisted steric stabilization of digestible lipids could also be explored to open new avenues in the field of lyotropic liquid crystalline phases. In a nutshell, the developed formulation strategy is well suited for improving the oral delivery of CoQ10 and could be explored for various difficultto-deliver drugs.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental methods; photograph of freeze dried CoQ10LCNPs; optical microscopic image of plain liquid crystalline phase, LCQ phase, PLCQ phase; chromatographic conditions for analysis of CoQ10; and time dependent accelerated stability of CoQ10-LCNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91172-2292055. Fax: +91172-2214692. E-mail: [email protected]; sanyogjain@rediffmail.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Director, NIPER for providing necessary infrastructure facilities. N.K.S. and K.T. are also thankful to Council of Scientific and Industrial Research (CSIR) for financial assistance, Government of India, New Delhi, India. Technical support rendered by Mr. Rahul Mahajan is duly acknowledged.

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REFERENCES

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