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A novel lipid nanoemulsion system for improved permeation of granisetron
Colloids and Surfaces B: Biointerfaces 101 (2013) 475–480
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A novel lipid nanoemulsion system for improved permeation of granisetron Hea-Jeong Doh a , Yunjin Jung a , Prabagar Balakrishnan b , Hyun-Jong Cho c,∗ , Dae-Duk Kim b,∗∗ a
College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea c College of Pharmacy, Sunchon National University, Sunchon 540-950, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 15 May 2012 Received in revised form 7 July 2012 Accepted 13 July 2012 Available online 21 July 2012 Keywords: Anti-emetics Caco-2 cell Enhanced permeation Granisetron Lipid nanoemulsion
a b s t r a c t A new lipid nanoemulsion (LNE) system containing granisetron (GRN) was developed and its in vitro permeation-enhancing effect was evaluated using Caco-2 cell monolayers. Particle size, polydispersity index (PI) and stability of the prepared GRN-loaded LNE systems were also characterized. The mean diameters of prepared LNEs were around 50 nm with PI < 0.2. Developed LNEs were stable at 4 ◦ C in the dark place over a period of 12 weeks. In vitro drug dissolution and cytotoxicity studies of GRN-loaded LNEs were performed. GRN-loaded LNEs exhibited significantly higher drug dissolution than GRN suspension at pH 6.8 for 2 h (P < 0.05). In vitro permeation study in Caco-2 cell monolayers showed that the LNEs significantly enhanced the drug permeation compared to GRN powder. The in vivo toxicity study in the rat jejunum revealed that the prepared GRN-loaded LNE was as safe as the commercial formulation (Kytril). These results suggest that LNE could be used as a potential oral liquid formulation of GRN for anti-emetic treatment on the post-operative and chemotherapeutic patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In recent years, much attention has focused on lipid-based formulations to improve the oral bioavailability of poorly watersoluble drugs. One of the most popular approaches is the incorporation of the active lipophilic component into the inert lipid vehicles such as oils, surfactant dispersions, microemulsions, nanoemulsions, self-emulsifying formulations, self-microemulsifying formulations, emulsions and liposomes [1–9]. Most of them can increase interfacial area of the drugs to improve solubilization behavior, as well as absorption across the mucosa. From the viewpoint of oral drug delivery, lipids can be investigated as components of various oily liquids and dispersions that are designed to improve solubility and bioavailability of drugs belonging to the class II and IV of the biopharmaceutical classification system [10]. One of the promising technologies for drug delivery is nanoemulsion system, which can be applied to enhance the oral bioavailability of the poorly water-soluble drugs. Nanoemulsion is known to be thermodynamically stable and transparent. It can be defined as a dispersion of oil and water stabilized by an interfacial film of surfactant molecules having the droplet size less than 100 nm [11]. Nanoemulsion can provide ultra low interfacial tensions and large o/w interfacial areas. It has a higher solubilization
∗ Corresponding author. Tel.: +82 61 750 3764; fax: +82 61 750 3708. ∗∗ Corresponding author. Tel.: +82 2 880 7870; fax: +82 2 873 9177. E-mail addresses: [email protected] (H.-J. Cho), [email protected] (D.-D. Kim). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.07.019
capacity than simple micellar solutions and its thermodynamic stability offers advantages over unstable dispersions, such as emulsions and suspensions, because it can be manufactured with little energy input (heat or mixing) and has a long shelf-life. The nanosized droplets leading to enormous interfacial areas associated with nanoemulsion would influence on the transport of the drug, an important factor in sustained and targeted drug delivery [12]. The attraction of formulating o/w nanoemulsion system lies in its ability to incorporate hydrophobic drugs into the oil phase, thereby enhancing their solubility [12]. It has also been reported that nanoemulsions can make bioavailability of drugs more reproducible [11,12]. Granisetron (GRN), {endo-1-methyl-N-[9-methyl-9azabicyclo(3,3,1)non-3-yl]-1H-indazo-le-3-carboxamide}, is one of the selective 5-HT3 receptor antagonists. Among 5-HT3 receptor antagonists, GRN may have less side effects, lower occurrence of drug–drug interaction and relatively longer half-life in the body [13]. It is reported that the selectivity of GRN to 5-HT3 receptors is 1000 times higher than that to the other serotonergic (5-HT1 , 5-HT1A , 5-HT1B , 5-HT1C , 5-HT2 ), ␣1 , ␣2 , -adrenergic, dopamine D2 , histamine H1 and opioid receptors, whereas it is 250–400:1 for ondansetron [14]. GRN is an effective and well-tolerated drug in the management of chemotherapy-induced, radiotherapy-induced and post-operative nausea and vomit in adults and children [15]. In particular, liquid formulations are usually regarded as the gold standard in the formulations especially for infants [16]. Moreover, people who have difficulty in swallowing would prefer liquid formulation than tablet [16]. It is well known that the oral
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absorption from liquid formulations is faster and higher than solid formulations. It is important in the treatment of anti-cancer therapy-induced nausea as it can provide faster relief to the distressed patients. GRN has also been available as GRN hydrochloride (HCl)-loaded oral solution in the market (Kytril, Roche, USA). However, several studies have reported that the oral absorption from nanoemulsion system can be enhanced compared to that of simple oral solution [17,18]. Thus, the objective of this study was to investigate the feasibility of a novel lipid nanoemulsion (LNE) system for oral delivery of GRN, for improving patient compliance, bioavailability and pharmacological efficacy. 2. Materials and methods 2.1. Materials GRN was kindly provided by Chong Kun Dang Pharm. Ltd. (Seoul, Korea). Egg phosphatidylcholine (Lipoid E80) was purchased from Lipoid AG (Ludwigshafen, Germany). Isopropyl myristate (IPM), hydroxypropylmethylcellulose (HPMC), hydroxypropyl-cyclodextrin (HP--CD) and poly(vinylpyrrolidone) (PVP) were purchased from Sigma Chemicals (St. Louis, MO, USA). Tween 80 and sodium taurocholate (NaTC) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Lauroglycol 90 (LG90), Labrafil and Labrafac were kindly gifted by Gattefosse (Cedex, France). Poloxamer 188 (F68) was obtained as a generous gift from BASF (Ludwigshafen, Germany). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All other chemicals were of analytical grade. 2.2. Solubility study The solubility of GRN in various oils was determined after adding an excess amount of drug in each 1 ml of selected oil (Table S1). The mixture was then kept at 25 ± 1.0 ◦ C in an isothermal shaker for 72 h to reach equilibrium state. The equilibrated samples were removed from shaker and centrifuged at 13,000 rpm for 15 min. The supernatant was taken and filtered through a 0.2-m membrane filter. The concentration of GRN in oils was then determined using high performance liquid chromatography (HPLC) system based on the reported method in our previous study [19]. It was equipped with a reverse phase C-18 column (XTerra, RP-18, 250 × 4.6 mm, 5 m, Waters Co., Milford, MA, USA), a pump (Waters 515), an automatic injector (Waters 717plus) and fluorescence detector (Series 200, PerkinElmer Instrument, Norwalk, CT, USA). The mobile phase was consisted of 25 mM acetate buffer (pH 4.8) and acetonitrile (72:28, v/v), and the eluent was monitored at 305 nm for excitation and 360 nm for emission with a flow rate of 1.0 ml/min. The injection volume for drug analysis was 50 l. The inter- and intra-day variance of this HPLC method was within the acceptable range. 2.3. Preparation of GRN-loaded LNEs GRN (0.4 mg/ml) and Lipoid E80 (1%, w/w) were dissolved in the oil phase (5%, w/w) composed of IPM and LG90 (1:1) at 70 ◦ C, and then stirred at the same temperature. Components such as F68 or HP-ß-CD or PVP (1%, w/w), Tween 80 (1%, w/w), glycerol (1%, w/w), HPMC (0.2%, w/w) and NaTC (1%, w/w) were added to deionized distilled water (DDW) at the equivalent temperature and stirring condition (Table S2). The oil phase was added drop-wise to the aqueous phase with constant stirring using a mechanical stirrer and the emulsion was allowed to form at the same temperature. The emulsion was then sonicated for 15 min using Vibra Cell VC750 probe-type sonicator (Sonics and Material Inc., Newtown, CT, USA)
at 21% of amplitude. The resulted LNEs were cooled down to the room temperature and stored at 4 ◦ C. 2.4. Characterizations of LNEs Mean diameters and polydispersity index (PI) values of oil droplets were measured by light scattering spectrophotometer ELS 8000 (Otsuka Electronics, Tokyo, Japan). The particle size was measured at 90◦ light scattering angle with intensity over 5000 during measurement. Morphology of LNE droplet was investigated with transmission electron microscopy (TEM) analysis (JEM 1010, JEOL, Tokyo, Japan). Approximately 20 l of LNE was diluted with water and stained by 2% (w/v) of phosphotungstic acid (PTA). And then it was placed on copper film grids and observed by TEM after drying in the air for 10 min. 2.5. Stability GRN incorporated in LNEs To identify GRN stability in LNE formulations during storage, the amount of GRN was analyzed quantitatively by HPLC over a period of 12 weeks at 4 ◦ C. At pre-determined times (0, 4, 8 and 12 weeks), each formulation was diluted with mobile phase and GRN content was analyzed by HPLC system. 2.6. In vitro dissolution study In vitro drug dissolution profiles from the prepared GRN-loaded LNEs and GRN powder were obtained using dialysis membrane (Viscase Corp., Chicago, IL, USA). Molecular weight cut-off of dialysis membrane was 12,000–14,000 Da. Aliquot (0.2 ml) of LNEs or GRN powder (suspended in phosphate buffered saline) was added in the dialysis bag, and these dialysis bags were incubated in the 100 ml of phosphate buffered saline (PBS, pH 6.8 adjusted with phosphoric acid) at 37 ◦ C. The speed of shaking bath was maintained as 100 rpm. Samples (1 ml each) were collected at determined times (15, 30, 45, 60, 90, 120, 180 and 240 min) and equivalent volume of fresh media (37 ◦ C) was replaced to maintain sink condition. The samples were filtered through 0.2-m membrane filter and analyzed by HPLC system for the determination of GRN content. 2.7. In vitro cytotoxicity test in Caco-2 cells The cytotoxicity of LNEs in Caco-2 cells was measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA). The CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). The human colon adenocarcinoma cell line (Caco-2) was obtained from the Korean Cell Line Bank (Seoul, Korea) and was grown in DMEM containing 10% FBS, 1% non-essential amino acids, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 ◦ C in the atmosphere of 5% CO2 and 95% relative humidity. When the Caco-2 cells (passage number: 24–25) acquired 70–80% confluency, the cells were detached from cell culture plate and seeded on 48-well plate at a density of 1.0 × 105 cells per well. After 24 h of incubation, the growth media were eliminated and 500 l of culture media containing different concentrations of formulations (0.5, 1 and 2%, v/v) was added to each well and incubated for 1 h at 37 ◦ C in the atmosphere of 5% CO2 and 95% relative humidity. Then, 1% (v/v) of each formulation was incubated for 1, 2 and 3 h at the same culture condition. After incubation, the Caco-2 cells were treated with 80 l of MTS-based CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent in 5% CO2 atmosphere condition at 37 ◦ C for 4 h. The absorbance
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was detected at 490 nm wavelength by EMax precision microplate reader (Molecular Devices, Sunnyvale, CA, USA). 2.8. In vitro drug permeation and post-cytotoxicity tests in Caco-2 cell monolayers In vitro permeation study in the Caco-2 cell monolayers was performed according to the modified reported method [4]. Briefly, when the Caco-2 cells acquired 70–80% confluency, the cells were trypsinized from the cell culture plate and seeded on collagencoated Transwell insert (0.4 m pore size, 12 mm membrane diameter, 12-well plate, Corning Costar Corp., Cambridge, MA, USA) at a density of 2.0 × 105 cells per well. In vitro absorption study was performed in the Transwell inserts when transepithelial electrical resistance (TEER) value was higher than 300 ohm cm2 measured by EVOM voltohmmeter (WPI, Sarasota, FL, USA). The culture media of Caco-2 cell monolayers were removed and cell monolayers were incubated with transport media, Hank’s balanced salt solution (HBSS) supplemented with 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 10 mM d-(+)-glucose, for 30 min at 37 ◦ C to get steady state for the transport study. The prepared 40 l of LNE mixed with 360 l of culture media or GRN powder in 400 l of culture media was loaded in the apical side of Transwell inserts. To calculate the permeated amounts of GRN from the apical side to the basolateral side (A to B), 1 ml was collected at pre-determined times (15, 30, 45, 60, 90 and 120 min) from basolateral side and replaced with the equivalent volume of fresh transport media. The permeated amounts of GRN were determined by HPLC method. After 2 h of transport study, Caco-2 cell viability (%) was measured by pre-described MTS-based assay. 2.9. In vivo toxicity test Approval for in vivo animal study was obtained from Seoul National University animal ethics committee and their guidelines were applied for the studies. Male Sprague-Dawley (SD) rats weighing about 300 g were fasted for 18 h before the experiments. They were anesthetized by intramusclar injection of ketamine and acepromazine mixture (4:1, v/v). Kytril tablet and prepared LNE (F3) were administered by oral route and GRN solution in PBS was injected into rat superficial femoral vein at 0.56 mg/kg dose. In the case of control group, no reagent was treated. The jejunum of rat was taken by excising the peritoneal membrane 8 h after drug administration. Excised jejunum was fixed in 10% (v/v) formaldehyde for at least 1 day. After fixation, tissues were rinsed in tap water and dehydrated using alcohol, then embedded in paraffin and sectioned into 5–10 m of thickness. The sections were treated with hematoxylin-eosin (H&E) staining for microscopic observation. 2.10. Data analysis All the experiments in the study were performed at least three times and the data were expressed as the mean ± standard deviation (SD). Statistical analysis of data was performed using analysis of variance. 3. Results and discussion 3.1. Preparation and characterization of GRN-loaded LNEs An important consideration for screening the components of nanoemulsion is that they should be pharmaceutically acceptable for oral administration. The higher solubility of the drug in the oil phase is important for the nanoemulsion to maintain the solubilized form of drug. Although the surfactant or cosurfactant can
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Table 1 Mean diameter and PI values of the developed GRN-loaded LNEs. Formulations
Mean diameter (nm, mean ± SD)
Polydispersity index (PI)
F1 F2 F3
57.00 ± 1.49 55.70 ± 0.70 46.77 ± 0.45
0.19 ± 0.01 0.17 ± 0.02 0.15 ± 0.03
contribute to the drug solubilization, dilution of nanoemulsion in the gastrointestinal tract may lead to the lowering of solvent capacity of surfactant or cosurfactant [4,6]. The process is thermodynamically driven by the requirement of the surfactant to maintain an aqueous phase concentration equivalent to its critical micelle concentration under the prevailing conditions of temperature, pH and ionic strength [20,23]. Thus, in the present study, oils from different categories and even mixture of oils were tested to select the highest solubility of GRN (Table S1). To prepare LNE system, the mixture of IPM and LG90 (1:1) was selected as it showed the highest drug solubility. Lipoid E-80, composed of mainly egg phosphatidylcholine, was used as the primary emulsifier for stabilization of the oil droplets. Manufacturer’s specification (Lipoid, Ludwigshafen, Germany) provides that Lipoid E-80 is a mixture of phospholipids from egg yolk sources with phosphatidylcholine (the major constituent) and a small fraction of zwitterionic and neutral phospholipids that would contribute a net negative charge at neutral pH [21]. Moreover, F68, HP--CD and PVP K25 were added as co-emulsifiers, which were reported for their solubilization and permeation-enhancing effect [22–24]. In addition, glycerol was added as a cosolvent in these nanoemulsion systems. HPMC was incorporated in all LNEs as a mucoadhesive polymer to increase the residence time in the intestinal membrane and to improve the drug absorption. NaTC (1%) was used as a surfactant as it has been reported as an absorption enhancer in the intestinal epithelial cell line [25]. Final GRN concentration in LNE systems was set up as 0.4 mg/ml in this study notwithstanding its improved GRN solubility due to LNE systems, considering its clinical dose (2 mg once daily or 1 mg twice daily) and oral administration volume. Three GRN-loaded LNE formulations (Table S2) were prepared in this study by the ultrasonication method. Ultrasound energy has been successfully applied in reducing the oil droplet size of the LNEs. In this study, the mean oil droplet diameters were around 50 nm (Table 1). PI values (<0.2) of the prepared formulations indicated narrow particle size distribution. Moreover, the PTA-stained oil droplets observed by TEM were round shape, indicating the formation of stable nanoemulsion. Particle sizes observed by TEM were coincided with those values measured by light scattering spectrophotometer (Fig. 1). In addition, the change of drug contents as a function of storage period was also measured. There was no significant change in drug contents in the prepared LNEs over a period of 12 weeks (Fig. 2). 3.2. In vitro dissolution study Drug dissolution studies were performed with the LNE formulations (Table S2) and drug dissolution profiles in PBS are presented in Fig. 3. GRN suspended in PBS was used as a control group. GRNloaded LNEs exhibited higher drug release profile compared to the control group. At the end of 4 h the drug release was >87%, whereas it was 68% in control. The higher drug release profile obtained from LNEs might be due to their smaller particle size and the higher drug solubilization capacity [6]. It appeared that the presence of different co-emulsifiers, such as F68, HP--CD, and PVP K-25, did not significantly influence on the GRN release pattern from LNE formulations. Moreover, in this dissolution study, dialysis membrane was
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Fig. 1. TEM images of prepared GRN-loaded LNEs: (A) F1, (B) F2, (C) F3. The length of bar is 200 nm.
3.3. In vitro cytotoxicity study in Caco-2 cells
140 0 week 4 weeks 8 weeks 12 weeks
Concentration- and incubation time-dependent cytotoxicity of GRN-LNE formulations was measured by MTS-based assay in Caco2 cells (Fig. 4). In GRN-LNE concentration-dependent study, three different volume ratio of GRN-LNE to Caco-2 cell culture media (0.5, 1 and 2%, v/v) was applied for 1 h. As shown in Fig. 4A, all
100
80
A
60
40
20
0 F1
F2
F3 ◦
Fig. 2. Stability test of GRN-loaded LNEs over 12 weeks in the dark place at 4 C. The change of GRN content (%) is shown. The data are expressed as the mean ± SD (n = 3).
Caco-2 cell viability (%)
GRN content (%)
120
120
100
80
60
40
20
0 F1
used to eliminate the possibility of the nanoemulsions dispersed in the release media, thereby ensuring more accurate dissolution profiles of the drug itself. Thus, it seems that the dialysis method may reflect the actual release profile of GRN from the LNEs [4].
Caco-2 cell viability (%)
B
Released amounts of GRN (%)
120
100
80
60
F2
F3
F2
F3
GRN-LNE (0.5%, 1h) GRN-LNE (1.0%, 1h) GRN-LNE (2.0%, 1h)
120
100
80
60
40
20
40 0
F1 F2 F3 GRN
20
F1 GRN-LNE (1h, 1%) GRN-LNE (2h, 1%) GRN-LNE (3h, 1%)
0 0
50
100
150
200
250
300
Time (min) Fig. 3. In vitro release profiles of GRN from various LNE formulations in PBS (pH 6.8) at 37 ◦ C. Each point indicates the mean ± SD (n = 3).
Fig. 4. GRN-loaded LNE concentration-dependent (A) and incubation timedependent (B) cytotoxicity tests in Caco-2 cells. Cell viability (%) is shown as the ratio to the control (no treatment) group. The data are expressed as the mean ± SD (n = 3).
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Caco-2 cell viability (%)
120 F1
100
F3
80 F2 GRN
60
40
20
0 0
50
100
150
200
250
300
Ratio of permeated GRN within 2 h (%) Fig. 5. In vitro permeation study in the Caco-2 cell monolayers. Correlationship between the ratio of permeated GRN from various formulations and Caco-2 cell viability after 2 h of transport study is exhibited (n = 3).
the LNE formulations showed higher Caco-2 cell viability and there was no significant difference among them. In the incubation timedependent cytotoxicity assay using 1% (v/v) of LNE to the cell culture media, it was observed that the cell viability decreased insignificantly as the incubation time increased (Fig. 4B). There was no significant difference observed in the Caco-2 cell viability values of the prepared LNEs. This result indicated that the prepared LNEs could be considerably safe to the epithelium of gastrointestinal tract after oral administration. 3.4. In vitro permeation study To predict the contribution of LNE system on the transport of GRN across the intestinal membrane, in vitro permeation study was performed in the Caco-2 cell monolayers. Moreover, to further establish the safety of the prepared LNEs, cytotoxicity studies were performed using MTS-based assay after 2 h of transport study in the Caco-2 cell monolayers. It was observed that all the LNEs exhibited higher drug permeation rates than that of GRN itself (Fig. 5). Among the LNEs tested, F3 showed the highest permeability. F3 showed 2.78 times higher drug permeation than that of GRN powder (P < 0.01). In general, the change of TEER value can be used as one of the indicators for the cytotoxicity in the Caco-2 cell monolayers. However, it is insufficient to determine the cytotoxic effect of formulations. Thus, MTS-based assay was performed to evaluate the cytotoxic effects of prepared LNE formulations on the Caco-2 cell monolayers during permeation study. To better understand the advantage of the prepared LNEs, the relationship between the Caco-2 cell viability and the permeated amount of GRN from different formulations was plotted (Fig. 5). It was observed that
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the cell viability and GRN permeation rate were higher in LNE formulations compared to the control group (Fig. 5). It can be also concluded that the type of emulsifier did not show any significant difference in drug permeation. The higher drug permeation from LNEs across Caco-2 cell monolayers could be explained by several mechanisms. It has been reported that microemulsion system can enhance the permeation of drug by opening the tight junction temporarily and facilitate drug transport via paracellular route [4,6]. It has been reported that the components of the microemulsion could also contribute to the enhanced drug transport across the mucosal membrane [4,26]. In this study, Lipoid E-80 was used as a surfactant and has been reported for its permeation-enhancing effect [21]. HP--CD and NaTC were also known for their permeation enhancing effect [27,28]. Moreover, it is assumed that the characteristics of LNE, such as small droplet size and the interaction between the surfactants and cellular membrane, may contribute to the permeation enhancement of GRN. In addition, mucoadhesiveness of HPMC may contribute to the increase of the residence time in the epithelium and enhance absorption across the mucosal membrane, consequently improving the bioavailability of drug [29,30]. It was also reported that lipid-based formulations can improve intestinal lymphatic transport of drug, thereby enhancing its oral bioavailability [31]. Although the lymphatic transport of GRN from developed LNEs should be further investigated, it has potential enhancement of bioavailability. 3.5. In vivo toxicity test of GRN-loaded LNEs In vivo toxicity of the prepared LNEs in rats was assessed by examining its intestine, especially jejunum except Peyer’s patch, after intravenous (IV) injection of GRN solution and oral administration of LNE (F3) and commercial formulation (Kytril tablet). The rat intestine was dissected and stained by H&E method. As shown in Fig. 6, intestinal epithelium, microvilli, junctions and mucosal structures were normal and there was no sign of inflammation and erosion in the control group. No pathological change was observed with the IV injection group. In the case of Kytril tablet and F3 formulation, ignorable pathological abnormalities, such as inflammation and erosion, were observed. There was no significant pathophysiological difference in the morphology between LNE and Kytril tablet-treated group. This result indicated that LNE (F3) could be safe as Kytril. However, specific investigation may be further required to determine the inflammation level with more elaborate manner. Whereas microemulsion systems are generally composed of large portions of oil phase and surfactants, the developed LNEs included less than 10.2% (w/w) of oil and surfactants. Thus, it was expected that they might have significantly lower cytotoxicity rather than typical microemulsion system and their safety was identified by in vitro and in vivo toxicity tests. Relatively lower toxicity of prepared LNE can be one of the merits along with its permeation-enhancing effects across intestinal epithelial cell
Fig. 6. In vivo toxicity tests by H&E staining in the rat jejunum. Microscopic images of (A) control group, (B) free GRN (intravenous injection), (C) Kytril tablet (oral administration), and (D) F3 (oral administration) are shown (×100).
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monolayers (Fig. 5). Considering all of these results, prepared LNE could be a potential oral liquid formulation for GRN delivery. 4. Conclusions In this study, GRN-loaded LNEs were developed using the mixture of IPM and LG90 as an oil phase, Lipoid E80 as an emulsifier, F68, HP--CD and PVP K25 as co-emulsifiers, glycerol as a cosolvent, HPMC as a mucoadhesive polymer, and NaTC as a permeation enhancer. The oil droplet size of prepared LNEs was around 50 nm. The LNEs exhibited higher drug dissolution profile than control group. The optimized LNE formulation showed low toxicity in Caco2 cells and in the jejunum of rat. The LNEs showed higher in vitro drug permeation rate than that of GRN in the Caco-2 cell monolayers. These results showed that the prepared LNE could be a potential liquid oral formulation of GRN in the anti-emetic treatment in the chemotherapeutic and post-operative patients. Acknowledgements This paper was supported by Sunchon National University Research Fund in 2012 and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0030635). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2012.07.019. References [1] V.J. Stella, J. Haslam, N. Yata, H. Okada, S. Lindenbaum, T. Higuchi, J. Pharm. Sci. 67 (1978) 1375.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A novel lipid nanoemulsion system for improved permeation of granisetron Hea-Jeong Doh a , Yunjin Jung a , Prabagar Balakrishnan b , Hyun-Jong Cho c,∗ , Dae-Duk Kim b,∗∗ a
College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea c College of Pharmacy, Sunchon National University, Sunchon 540-950, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 15 May 2012 Received in revised form 7 July 2012 Accepted 13 July 2012 Available online 21 July 2012 Keywords: Anti-emetics Caco-2 cell Enhanced permeation Granisetron Lipid nanoemulsion
a b s t r a c t A new lipid nanoemulsion (LNE) system containing granisetron (GRN) was developed and its in vitro permeation-enhancing effect was evaluated using Caco-2 cell monolayers. Particle size, polydispersity index (PI) and stability of the prepared GRN-loaded LNE systems were also characterized. The mean diameters of prepared LNEs were around 50 nm with PI < 0.2. Developed LNEs were stable at 4 ◦ C in the dark place over a period of 12 weeks. In vitro drug dissolution and cytotoxicity studies of GRN-loaded LNEs were performed. GRN-loaded LNEs exhibited significantly higher drug dissolution than GRN suspension at pH 6.8 for 2 h (P < 0.05). In vitro permeation study in Caco-2 cell monolayers showed that the LNEs significantly enhanced the drug permeation compared to GRN powder. The in vivo toxicity study in the rat jejunum revealed that the prepared GRN-loaded LNE was as safe as the commercial formulation (Kytril). These results suggest that LNE could be used as a potential oral liquid formulation of GRN for anti-emetic treatment on the post-operative and chemotherapeutic patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In recent years, much attention has focused on lipid-based formulations to improve the oral bioavailability of poorly watersoluble drugs. One of the most popular approaches is the incorporation of the active lipophilic component into the inert lipid vehicles such as oils, surfactant dispersions, microemulsions, nanoemulsions, self-emulsifying formulations, self-microemulsifying formulations, emulsions and liposomes [1–9]. Most of them can increase interfacial area of the drugs to improve solubilization behavior, as well as absorption across the mucosa. From the viewpoint of oral drug delivery, lipids can be investigated as components of various oily liquids and dispersions that are designed to improve solubility and bioavailability of drugs belonging to the class II and IV of the biopharmaceutical classification system [10]. One of the promising technologies for drug delivery is nanoemulsion system, which can be applied to enhance the oral bioavailability of the poorly water-soluble drugs. Nanoemulsion is known to be thermodynamically stable and transparent. It can be defined as a dispersion of oil and water stabilized by an interfacial film of surfactant molecules having the droplet size less than 100 nm [11]. Nanoemulsion can provide ultra low interfacial tensions and large o/w interfacial areas. It has a higher solubilization
∗ Corresponding author. Tel.: +82 61 750 3764; fax: +82 61 750 3708. ∗∗ Corresponding author. Tel.: +82 2 880 7870; fax: +82 2 873 9177. E-mail addresses: [email protected] (H.-J. Cho), [email protected] (D.-D. Kim). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.07.019
capacity than simple micellar solutions and its thermodynamic stability offers advantages over unstable dispersions, such as emulsions and suspensions, because it can be manufactured with little energy input (heat or mixing) and has a long shelf-life. The nanosized droplets leading to enormous interfacial areas associated with nanoemulsion would influence on the transport of the drug, an important factor in sustained and targeted drug delivery [12]. The attraction of formulating o/w nanoemulsion system lies in its ability to incorporate hydrophobic drugs into the oil phase, thereby enhancing their solubility [12]. It has also been reported that nanoemulsions can make bioavailability of drugs more reproducible [11,12]. Granisetron (GRN), {endo-1-methyl-N-[9-methyl-9azabicyclo(3,3,1)non-3-yl]-1H-indazo-le-3-carboxamide}, is one of the selective 5-HT3 receptor antagonists. Among 5-HT3 receptor antagonists, GRN may have less side effects, lower occurrence of drug–drug interaction and relatively longer half-life in the body [13]. It is reported that the selectivity of GRN to 5-HT3 receptors is 1000 times higher than that to the other serotonergic (5-HT1 , 5-HT1A , 5-HT1B , 5-HT1C , 5-HT2 ), ␣1 , ␣2 , -adrenergic, dopamine D2 , histamine H1 and opioid receptors, whereas it is 250–400:1 for ondansetron [14]. GRN is an effective and well-tolerated drug in the management of chemotherapy-induced, radiotherapy-induced and post-operative nausea and vomit in adults and children [15]. In particular, liquid formulations are usually regarded as the gold standard in the formulations especially for infants [16]. Moreover, people who have difficulty in swallowing would prefer liquid formulation than tablet [16]. It is well known that the oral
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absorption from liquid formulations is faster and higher than solid formulations. It is important in the treatment of anti-cancer therapy-induced nausea as it can provide faster relief to the distressed patients. GRN has also been available as GRN hydrochloride (HCl)-loaded oral solution in the market (Kytril, Roche, USA). However, several studies have reported that the oral absorption from nanoemulsion system can be enhanced compared to that of simple oral solution [17,18]. Thus, the objective of this study was to investigate the feasibility of a novel lipid nanoemulsion (LNE) system for oral delivery of GRN, for improving patient compliance, bioavailability and pharmacological efficacy. 2. Materials and methods 2.1. Materials GRN was kindly provided by Chong Kun Dang Pharm. Ltd. (Seoul, Korea). Egg phosphatidylcholine (Lipoid E80) was purchased from Lipoid AG (Ludwigshafen, Germany). Isopropyl myristate (IPM), hydroxypropylmethylcellulose (HPMC), hydroxypropyl-cyclodextrin (HP--CD) and poly(vinylpyrrolidone) (PVP) were purchased from Sigma Chemicals (St. Louis, MO, USA). Tween 80 and sodium taurocholate (NaTC) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Lauroglycol 90 (LG90), Labrafil and Labrafac were kindly gifted by Gattefosse (Cedex, France). Poloxamer 188 (F68) was obtained as a generous gift from BASF (Ludwigshafen, Germany). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All other chemicals were of analytical grade. 2.2. Solubility study The solubility of GRN in various oils was determined after adding an excess amount of drug in each 1 ml of selected oil (Table S1). The mixture was then kept at 25 ± 1.0 ◦ C in an isothermal shaker for 72 h to reach equilibrium state. The equilibrated samples were removed from shaker and centrifuged at 13,000 rpm for 15 min. The supernatant was taken and filtered through a 0.2-m membrane filter. The concentration of GRN in oils was then determined using high performance liquid chromatography (HPLC) system based on the reported method in our previous study [19]. It was equipped with a reverse phase C-18 column (XTerra, RP-18, 250 × 4.6 mm, 5 m, Waters Co., Milford, MA, USA), a pump (Waters 515), an automatic injector (Waters 717plus) and fluorescence detector (Series 200, PerkinElmer Instrument, Norwalk, CT, USA). The mobile phase was consisted of 25 mM acetate buffer (pH 4.8) and acetonitrile (72:28, v/v), and the eluent was monitored at 305 nm for excitation and 360 nm for emission with a flow rate of 1.0 ml/min. The injection volume for drug analysis was 50 l. The inter- and intra-day variance of this HPLC method was within the acceptable range. 2.3. Preparation of GRN-loaded LNEs GRN (0.4 mg/ml) and Lipoid E80 (1%, w/w) were dissolved in the oil phase (5%, w/w) composed of IPM and LG90 (1:1) at 70 ◦ C, and then stirred at the same temperature. Components such as F68 or HP-ß-CD or PVP (1%, w/w), Tween 80 (1%, w/w), glycerol (1%, w/w), HPMC (0.2%, w/w) and NaTC (1%, w/w) were added to deionized distilled water (DDW) at the equivalent temperature and stirring condition (Table S2). The oil phase was added drop-wise to the aqueous phase with constant stirring using a mechanical stirrer and the emulsion was allowed to form at the same temperature. The emulsion was then sonicated for 15 min using Vibra Cell VC750 probe-type sonicator (Sonics and Material Inc., Newtown, CT, USA)
at 21% of amplitude. The resulted LNEs were cooled down to the room temperature and stored at 4 ◦ C. 2.4. Characterizations of LNEs Mean diameters and polydispersity index (PI) values of oil droplets were measured by light scattering spectrophotometer ELS 8000 (Otsuka Electronics, Tokyo, Japan). The particle size was measured at 90◦ light scattering angle with intensity over 5000 during measurement. Morphology of LNE droplet was investigated with transmission electron microscopy (TEM) analysis (JEM 1010, JEOL, Tokyo, Japan). Approximately 20 l of LNE was diluted with water and stained by 2% (w/v) of phosphotungstic acid (PTA). And then it was placed on copper film grids and observed by TEM after drying in the air for 10 min. 2.5. Stability GRN incorporated in LNEs To identify GRN stability in LNE formulations during storage, the amount of GRN was analyzed quantitatively by HPLC over a period of 12 weeks at 4 ◦ C. At pre-determined times (0, 4, 8 and 12 weeks), each formulation was diluted with mobile phase and GRN content was analyzed by HPLC system. 2.6. In vitro dissolution study In vitro drug dissolution profiles from the prepared GRN-loaded LNEs and GRN powder were obtained using dialysis membrane (Viscase Corp., Chicago, IL, USA). Molecular weight cut-off of dialysis membrane was 12,000–14,000 Da. Aliquot (0.2 ml) of LNEs or GRN powder (suspended in phosphate buffered saline) was added in the dialysis bag, and these dialysis bags were incubated in the 100 ml of phosphate buffered saline (PBS, pH 6.8 adjusted with phosphoric acid) at 37 ◦ C. The speed of shaking bath was maintained as 100 rpm. Samples (1 ml each) were collected at determined times (15, 30, 45, 60, 90, 120, 180 and 240 min) and equivalent volume of fresh media (37 ◦ C) was replaced to maintain sink condition. The samples were filtered through 0.2-m membrane filter and analyzed by HPLC system for the determination of GRN content. 2.7. In vitro cytotoxicity test in Caco-2 cells The cytotoxicity of LNEs in Caco-2 cells was measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA). The CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). The human colon adenocarcinoma cell line (Caco-2) was obtained from the Korean Cell Line Bank (Seoul, Korea) and was grown in DMEM containing 10% FBS, 1% non-essential amino acids, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 ◦ C in the atmosphere of 5% CO2 and 95% relative humidity. When the Caco-2 cells (passage number: 24–25) acquired 70–80% confluency, the cells were detached from cell culture plate and seeded on 48-well plate at a density of 1.0 × 105 cells per well. After 24 h of incubation, the growth media were eliminated and 500 l of culture media containing different concentrations of formulations (0.5, 1 and 2%, v/v) was added to each well and incubated for 1 h at 37 ◦ C in the atmosphere of 5% CO2 and 95% relative humidity. Then, 1% (v/v) of each formulation was incubated for 1, 2 and 3 h at the same culture condition. After incubation, the Caco-2 cells were treated with 80 l of MTS-based CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent in 5% CO2 atmosphere condition at 37 ◦ C for 4 h. The absorbance
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was detected at 490 nm wavelength by EMax precision microplate reader (Molecular Devices, Sunnyvale, CA, USA). 2.8. In vitro drug permeation and post-cytotoxicity tests in Caco-2 cell monolayers In vitro permeation study in the Caco-2 cell monolayers was performed according to the modified reported method [4]. Briefly, when the Caco-2 cells acquired 70–80% confluency, the cells were trypsinized from the cell culture plate and seeded on collagencoated Transwell insert (0.4 m pore size, 12 mm membrane diameter, 12-well plate, Corning Costar Corp., Cambridge, MA, USA) at a density of 2.0 × 105 cells per well. In vitro absorption study was performed in the Transwell inserts when transepithelial electrical resistance (TEER) value was higher than 300 ohm cm2 measured by EVOM voltohmmeter (WPI, Sarasota, FL, USA). The culture media of Caco-2 cell monolayers were removed and cell monolayers were incubated with transport media, Hank’s balanced salt solution (HBSS) supplemented with 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 10 mM d-(+)-glucose, for 30 min at 37 ◦ C to get steady state for the transport study. The prepared 40 l of LNE mixed with 360 l of culture media or GRN powder in 400 l of culture media was loaded in the apical side of Transwell inserts. To calculate the permeated amounts of GRN from the apical side to the basolateral side (A to B), 1 ml was collected at pre-determined times (15, 30, 45, 60, 90 and 120 min) from basolateral side and replaced with the equivalent volume of fresh transport media. The permeated amounts of GRN were determined by HPLC method. After 2 h of transport study, Caco-2 cell viability (%) was measured by pre-described MTS-based assay. 2.9. In vivo toxicity test Approval for in vivo animal study was obtained from Seoul National University animal ethics committee and their guidelines were applied for the studies. Male Sprague-Dawley (SD) rats weighing about 300 g were fasted for 18 h before the experiments. They were anesthetized by intramusclar injection of ketamine and acepromazine mixture (4:1, v/v). Kytril tablet and prepared LNE (F3) were administered by oral route and GRN solution in PBS was injected into rat superficial femoral vein at 0.56 mg/kg dose. In the case of control group, no reagent was treated. The jejunum of rat was taken by excising the peritoneal membrane 8 h after drug administration. Excised jejunum was fixed in 10% (v/v) formaldehyde for at least 1 day. After fixation, tissues were rinsed in tap water and dehydrated using alcohol, then embedded in paraffin and sectioned into 5–10 m of thickness. The sections were treated with hematoxylin-eosin (H&E) staining for microscopic observation. 2.10. Data analysis All the experiments in the study were performed at least three times and the data were expressed as the mean ± standard deviation (SD). Statistical analysis of data was performed using analysis of variance. 3. Results and discussion 3.1. Preparation and characterization of GRN-loaded LNEs An important consideration for screening the components of nanoemulsion is that they should be pharmaceutically acceptable for oral administration. The higher solubility of the drug in the oil phase is important for the nanoemulsion to maintain the solubilized form of drug. Although the surfactant or cosurfactant can
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Table 1 Mean diameter and PI values of the developed GRN-loaded LNEs. Formulations
Mean diameter (nm, mean ± SD)
Polydispersity index (PI)
F1 F2 F3
57.00 ± 1.49 55.70 ± 0.70 46.77 ± 0.45
0.19 ± 0.01 0.17 ± 0.02 0.15 ± 0.03
contribute to the drug solubilization, dilution of nanoemulsion in the gastrointestinal tract may lead to the lowering of solvent capacity of surfactant or cosurfactant [4,6]. The process is thermodynamically driven by the requirement of the surfactant to maintain an aqueous phase concentration equivalent to its critical micelle concentration under the prevailing conditions of temperature, pH and ionic strength [20,23]. Thus, in the present study, oils from different categories and even mixture of oils were tested to select the highest solubility of GRN (Table S1). To prepare LNE system, the mixture of IPM and LG90 (1:1) was selected as it showed the highest drug solubility. Lipoid E-80, composed of mainly egg phosphatidylcholine, was used as the primary emulsifier for stabilization of the oil droplets. Manufacturer’s specification (Lipoid, Ludwigshafen, Germany) provides that Lipoid E-80 is a mixture of phospholipids from egg yolk sources with phosphatidylcholine (the major constituent) and a small fraction of zwitterionic and neutral phospholipids that would contribute a net negative charge at neutral pH [21]. Moreover, F68, HP--CD and PVP K25 were added as co-emulsifiers, which were reported for their solubilization and permeation-enhancing effect [22–24]. In addition, glycerol was added as a cosolvent in these nanoemulsion systems. HPMC was incorporated in all LNEs as a mucoadhesive polymer to increase the residence time in the intestinal membrane and to improve the drug absorption. NaTC (1%) was used as a surfactant as it has been reported as an absorption enhancer in the intestinal epithelial cell line [25]. Final GRN concentration in LNE systems was set up as 0.4 mg/ml in this study notwithstanding its improved GRN solubility due to LNE systems, considering its clinical dose (2 mg once daily or 1 mg twice daily) and oral administration volume. Three GRN-loaded LNE formulations (Table S2) were prepared in this study by the ultrasonication method. Ultrasound energy has been successfully applied in reducing the oil droplet size of the LNEs. In this study, the mean oil droplet diameters were around 50 nm (Table 1). PI values (<0.2) of the prepared formulations indicated narrow particle size distribution. Moreover, the PTA-stained oil droplets observed by TEM were round shape, indicating the formation of stable nanoemulsion. Particle sizes observed by TEM were coincided with those values measured by light scattering spectrophotometer (Fig. 1). In addition, the change of drug contents as a function of storage period was also measured. There was no significant change in drug contents in the prepared LNEs over a period of 12 weeks (Fig. 2). 3.2. In vitro dissolution study Drug dissolution studies were performed with the LNE formulations (Table S2) and drug dissolution profiles in PBS are presented in Fig. 3. GRN suspended in PBS was used as a control group. GRNloaded LNEs exhibited higher drug release profile compared to the control group. At the end of 4 h the drug release was >87%, whereas it was 68% in control. The higher drug release profile obtained from LNEs might be due to their smaller particle size and the higher drug solubilization capacity [6]. It appeared that the presence of different co-emulsifiers, such as F68, HP--CD, and PVP K-25, did not significantly influence on the GRN release pattern from LNE formulations. Moreover, in this dissolution study, dialysis membrane was
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Fig. 1. TEM images of prepared GRN-loaded LNEs: (A) F1, (B) F2, (C) F3. The length of bar is 200 nm.
3.3. In vitro cytotoxicity study in Caco-2 cells
140 0 week 4 weeks 8 weeks 12 weeks
Concentration- and incubation time-dependent cytotoxicity of GRN-LNE formulations was measured by MTS-based assay in Caco2 cells (Fig. 4). In GRN-LNE concentration-dependent study, three different volume ratio of GRN-LNE to Caco-2 cell culture media (0.5, 1 and 2%, v/v) was applied for 1 h. As shown in Fig. 4A, all
100
80
A
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40
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0 F1
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Fig. 2. Stability test of GRN-loaded LNEs over 12 weeks in the dark place at 4 C. The change of GRN content (%) is shown. The data are expressed as the mean ± SD (n = 3).
Caco-2 cell viability (%)
GRN content (%)
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used to eliminate the possibility of the nanoemulsions dispersed in the release media, thereby ensuring more accurate dissolution profiles of the drug itself. Thus, it seems that the dialysis method may reflect the actual release profile of GRN from the LNEs [4].
Caco-2 cell viability (%)
B
Released amounts of GRN (%)
120
100
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60
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F3
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GRN-LNE (0.5%, 1h) GRN-LNE (1.0%, 1h) GRN-LNE (2.0%, 1h)
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40 0
F1 F2 F3 GRN
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F1 GRN-LNE (1h, 1%) GRN-LNE (2h, 1%) GRN-LNE (3h, 1%)
0 0
50
100
150
200
250
300
Time (min) Fig. 3. In vitro release profiles of GRN from various LNE formulations in PBS (pH 6.8) at 37 ◦ C. Each point indicates the mean ± SD (n = 3).
Fig. 4. GRN-loaded LNE concentration-dependent (A) and incubation timedependent (B) cytotoxicity tests in Caco-2 cells. Cell viability (%) is shown as the ratio to the control (no treatment) group. The data are expressed as the mean ± SD (n = 3).
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Caco-2 cell viability (%)
120 F1
100
F3
80 F2 GRN
60
40
20
0 0
50
100
150
200
250
300
Ratio of permeated GRN within 2 h (%) Fig. 5. In vitro permeation study in the Caco-2 cell monolayers. Correlationship between the ratio of permeated GRN from various formulations and Caco-2 cell viability after 2 h of transport study is exhibited (n = 3).
the LNE formulations showed higher Caco-2 cell viability and there was no significant difference among them. In the incubation timedependent cytotoxicity assay using 1% (v/v) of LNE to the cell culture media, it was observed that the cell viability decreased insignificantly as the incubation time increased (Fig. 4B). There was no significant difference observed in the Caco-2 cell viability values of the prepared LNEs. This result indicated that the prepared LNEs could be considerably safe to the epithelium of gastrointestinal tract after oral administration. 3.4. In vitro permeation study To predict the contribution of LNE system on the transport of GRN across the intestinal membrane, in vitro permeation study was performed in the Caco-2 cell monolayers. Moreover, to further establish the safety of the prepared LNEs, cytotoxicity studies were performed using MTS-based assay after 2 h of transport study in the Caco-2 cell monolayers. It was observed that all the LNEs exhibited higher drug permeation rates than that of GRN itself (Fig. 5). Among the LNEs tested, F3 showed the highest permeability. F3 showed 2.78 times higher drug permeation than that of GRN powder (P < 0.01). In general, the change of TEER value can be used as one of the indicators for the cytotoxicity in the Caco-2 cell monolayers. However, it is insufficient to determine the cytotoxic effect of formulations. Thus, MTS-based assay was performed to evaluate the cytotoxic effects of prepared LNE formulations on the Caco-2 cell monolayers during permeation study. To better understand the advantage of the prepared LNEs, the relationship between the Caco-2 cell viability and the permeated amount of GRN from different formulations was plotted (Fig. 5). It was observed that
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the cell viability and GRN permeation rate were higher in LNE formulations compared to the control group (Fig. 5). It can be also concluded that the type of emulsifier did not show any significant difference in drug permeation. The higher drug permeation from LNEs across Caco-2 cell monolayers could be explained by several mechanisms. It has been reported that microemulsion system can enhance the permeation of drug by opening the tight junction temporarily and facilitate drug transport via paracellular route [4,6]. It has been reported that the components of the microemulsion could also contribute to the enhanced drug transport across the mucosal membrane [4,26]. In this study, Lipoid E-80 was used as a surfactant and has been reported for its permeation-enhancing effect [21]. HP--CD and NaTC were also known for their permeation enhancing effect [27,28]. Moreover, it is assumed that the characteristics of LNE, such as small droplet size and the interaction between the surfactants and cellular membrane, may contribute to the permeation enhancement of GRN. In addition, mucoadhesiveness of HPMC may contribute to the increase of the residence time in the epithelium and enhance absorption across the mucosal membrane, consequently improving the bioavailability of drug [29,30]. It was also reported that lipid-based formulations can improve intestinal lymphatic transport of drug, thereby enhancing its oral bioavailability [31]. Although the lymphatic transport of GRN from developed LNEs should be further investigated, it has potential enhancement of bioavailability. 3.5. In vivo toxicity test of GRN-loaded LNEs In vivo toxicity of the prepared LNEs in rats was assessed by examining its intestine, especially jejunum except Peyer’s patch, after intravenous (IV) injection of GRN solution and oral administration of LNE (F3) and commercial formulation (Kytril tablet). The rat intestine was dissected and stained by H&E method. As shown in Fig. 6, intestinal epithelium, microvilli, junctions and mucosal structures were normal and there was no sign of inflammation and erosion in the control group. No pathological change was observed with the IV injection group. In the case of Kytril tablet and F3 formulation, ignorable pathological abnormalities, such as inflammation and erosion, were observed. There was no significant pathophysiological difference in the morphology between LNE and Kytril tablet-treated group. This result indicated that LNE (F3) could be safe as Kytril. However, specific investigation may be further required to determine the inflammation level with more elaborate manner. Whereas microemulsion systems are generally composed of large portions of oil phase and surfactants, the developed LNEs included less than 10.2% (w/w) of oil and surfactants. Thus, it was expected that they might have significantly lower cytotoxicity rather than typical microemulsion system and their safety was identified by in vitro and in vivo toxicity tests. Relatively lower toxicity of prepared LNE can be one of the merits along with its permeation-enhancing effects across intestinal epithelial cell
Fig. 6. In vivo toxicity tests by H&E staining in the rat jejunum. Microscopic images of (A) control group, (B) free GRN (intravenous injection), (C) Kytril tablet (oral administration), and (D) F3 (oral administration) are shown (×100).
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monolayers (Fig. 5). Considering all of these results, prepared LNE could be a potential oral liquid formulation for GRN delivery. 4. Conclusions In this study, GRN-loaded LNEs were developed using the mixture of IPM and LG90 as an oil phase, Lipoid E80 as an emulsifier, F68, HP--CD and PVP K25 as co-emulsifiers, glycerol as a cosolvent, HPMC as a mucoadhesive polymer, and NaTC as a permeation enhancer. The oil droplet size of prepared LNEs was around 50 nm. The LNEs exhibited higher drug dissolution profile than control group. The optimized LNE formulation showed low toxicity in Caco2 cells and in the jejunum of rat. The LNEs showed higher in vitro drug permeation rate than that of GRN in the Caco-2 cell monolayers. These results showed that the prepared LNE could be a potential liquid oral formulation of GRN in the anti-emetic treatment in the chemotherapeutic and post-operative patients. Acknowledgements This paper was supported by Sunchon National University Research Fund in 2012 and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0030635). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2012.07.019. References [1] V.J. Stella, J. Haslam, N. Yata, H. Okada, S. Lindenbaum, T. Higuchi, J. Pharm. Sci. 67 (1978) 1375.
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