http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(4): 382–389 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2013.863396
ORIGINAL ARTICLE
Pharmacokinetics, tissue distribution and relative bioavailability of geniposide-solid lipid nanoparticles following oral administration Fugang Wang, Juan Cao, Jifu Hao, and Ke Liu
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School of Pharmacy, Taishan Medical University, Taian, China
Abstract
Keywords
Geniposide has various pharmacological effects; however, low oral bioavailability limits its clinical utility. This study explores the pharmacokinetics, tissue distribution and relative bioavailability of geniposide-solid lipid nanoparticles (SLNs) following oral administration. The geniposide solution and geniposide-SLNs were orally administered to the rats, respectively. The Cmax value of geniposide in the geniposide-SLNs was significantly higher than that obtained with geniposide solution. Compared with the geniposide solution, the t1/2 and MRT were prolonged; the CL and V1/F were increased with geniposide-SLNs. The AUC0–1values of geniposide-SLNs were 50 times greater than geniposide solution. The ratios of AUC0–8 h in the liver, spleen, heart, kidney, brain and lung of the geniposide-SLNs to geniposide solution were 25.93, 4.28, 27.91, 10.15, 5.16 and 16.22, respectively. Prepared geniposide-SLNs are very helpful for increasing the bioavailability of geniposide. These data suggest that SLNs are a promising delivery system to enhance the oral bioavailability of geniposide.
Bioavailability, geniposide, geniposide-SLNs, pharmacokinetics, tissue distribution
Introduction Geniposide (structure shown in Figure 1) is a major active ingredient in the traditional Chinese medicine, Fructus Gardeniae, which comes from the Gardenia jasminoides ELLIS fruits. Fructus Gardeniae is a popular Chinese herb for the treatment of febrile diseases including jaundice, acute conjunctivitis, epistaxis, hematemesis, hematuria, pyogenic infections and ulcers of the skin Chang and But, 1987). Various beneficial activities of geniposide have been reported, including antitumour (Hsu et al., 1997; Koo et al., 2004), anti-inflammation (Liaw and Chao, 2001; Koo et al., 2006), anti-thrombosis (Suzuki et al., 2001), immunosuppression (Chang et al., 2005), neuroprotection (Lee et al., 2006; Liu et al., 2009), hypoglycemic effect (Wu et al., 2009; Kojima et al., 2011) and promoting cell proliferation (Chen et al., 2010). Although geniposide has various protective effects, owing to the short elimination half-life, it may possibly lead to severe and acute side effects (Somers et al., 2008) because venous administration of frequent and high doses may be needed. But, for the treatment of chronic diseases, oral administration is the most preferred route regarding the conventional drug delivery system. However, the absorption of geniposide in vivo is very poor after oral administration in our previous study, which diminishes its therapeutic effects. Geniposide is soluble in water, but the low permeability making it a potential candidate for delivery via lipidbased nanoparticulate system. As this would not only improve gut permeability, but may also avoid hepatic first pass metabolism of geniposide. This will reduce both the therapeutic dose and side
Address for correspondence: Dr Fugang Wang, School of Pharmacy, Taishan Medical University, Changcheng Road, Taian, Shandong 271016, China. Tel: +86 538 6229751. Fax: +86 538 6229751. E-mail: [email protected]
History Received 12 May 2013 Revised 10 September 2013 Accepted 28 October 2013 Published online 1 April 2014
effects associated with long term use of geniposide. Thus, oral formulation with improved absorption of geniposide is highly desired. To improve the bioavailability of geniposide, we are using the novel drug delivery systems to improve the current drug therapy. Solid lipid nanoparticles (SLNs) are prepared from solid physiological lipids, resulting in a solid particle matrix that can be loaded with variety of drugs (Luo et al., 2011). SLNs can be prepared from biodegradable, fatty acids, triglycerides and phospholipids and so on, which are normal constituents of the human body (Westesen et al., 1997; Rawat et al., 2008; Souto and Mu¨ller, 2010). However, it is a major challenge for encapsulation of hydrophilic materials into the hydrophobic matrix of SLNs (Cortesi et al., 2002). We have found a method to prepare SNPs of hydrophilic drugs with improved entrapment efficiency. In this study, the primary aim is to investigate the pharmacokinetics of geniposide-SLNs after oral administration to the rats in improving the bioavailability of geniposide, and evaluate the tissue distribution (heart, liver, spleen, lung, brain and kidney) of geniposide-SLNs after oral administration to rats.
Materials and methods Materials Geniposide (purity 99%) was purchased from Ci Yuan Biotechnology Co., Ltd (Shanxi, China). Hyperoside (internal standard) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol and acetonitrile were the products of Merck (Darmstadt, Germany). All other chemicals were of analytical grade. SpragueDawley (SD) rats were obtained from Laboratory Animal Center of Shandong University of Tradition Chinese Medicine (Shandong, China). All animal handling and treatments followed the EC Directive 86/609/EEC.
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were the weight of drug added in the system and the weight of lipid added in the system, respectively. The entrapment efficiency (EE) and drug loading (DL) of geniposide-SLNs were calculated according to Equations (1) and (2): W2 W1 100 W2
ð1Þ
W2 W1 100 W2 W1 þ W3
ð2Þ
EEð%Þ ¼
DLð%Þ ¼
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In vitro release studies
Figure 1. Chemical structures of geniposide (MW404).
Preparation of geniposide-SLNs Solid lipid nanoparticles of geniposide were prepared following melt-emulsion ultrasonication method (Arai et al., 2009). The lipidic phase (glyceryl monostearate, geniposide and appropriate ethanol) and the aqueous phase (poloxamer 188 and water) were heated to 10 C above the lipid melt temperature, and the formulation composition had a geniposide/glyceryl monostearate/poloxamer 188 ratio of 0.3/1/1. Then, the organic phase was added into the hot aqueous phase under mechanical agitation. The resulting solution was kept at the same temperature with the same agitation speed. The proportion of two phases formed a microemulsion spontaneously. Hot microemulsion was treated for 5 minutes (work 2 seconds and stand 3 seconds) by a Lab ultrasonic cell pulveriser (JY92-II, Ningbo Scientz Biotechnology Co., Ltd, China) at 600 W, and rapidly cooled by immersing the beaker into ice water (0 C). The prepared geniposide-SLNs were used as such for further studies. Particle size, zeta potential and surface morphology of geniposide-SLNs Particle size and zeta potential were determined by Zetasizer NANO ZS (Malvern, England). To study the morphology like sphericity and aggregation, the sample was examined by Inspect F50 (FEI Co., Ltd, Hillsboro, OR). Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) was performed using a differential scanning calorimeter (DSC-60WS; Shimadzu, Japan). The following samples were placed in aluminium pans under a nitrogen purge for analysis: geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188, glyceryl monostearate. A scan rate of 10 C/minute was used over a temperature range of 35–200 C. Entrapment efficiency and drug loading determination) Geniposide-SLNs was precipitated by adding saturated (NH4)2SO4 solution, centrifugation (CS120GXL, Hitachi, Japan) at 50 000 rpm for 15 minutes, and then collecting the supernatant and solid residue. The solid residue was dispersed in 40 mL phosphate buffered saline (PBS) solution (pH 7.4), and then separated by centrifuge method. Both the drug content in supernatant and that in PBS were regarded as W1. W2 and W3
In vitro release was determined in phosphate buffer (pH 6.8) by dialysis bag method using dialysis membrane with a molecular of 12 000–14 000 Da. Two millilitre of geniposide-SLNs dispersion was placed inside the dialysis bag, tied at both the ends and placed into the preheated dissolution media (40 mL). Stirring was maintained at 100 rpm, a RCZ-8A Drug Dissolution Tester (Tianjin Medical Instrument Factory, China), at 37 0.5 C. Two millilitres aliquots were withdrawn at pre-set time intervals (0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h) and replaced by an equal volume of fresh dissolution medium. After suitable dilution, the samples were analysed by HPLC. Corrected concentration of geniposide was calculated in the test samples using the regression equation of the calibration curve. Drug administration and sampling Healthy Sprague-Dawley rats (weighing 250–300 g) were used and they were fasted overnight with free access to water. Twelve rats were divided into two groups: the geniposide-SLNs group and geniposide solution group. In the former group, animals were administered a single dose of geniposide-SLNs by gastric infusion (equivalent to 100 mg/kg of geniposide). In the latter group, those were administered 100 mg/kg of geniposide suspended in normal saline. Blood samples (0.4 mL) were collected orbital venous at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h after dosing. Heparin was used as an anticoagulant. The blood samples were centrifuged at 10 000 g for 10 minutes to separate the plasma and stored at 20 C until analysis. For the tissue distribution, 60 rats received the same dosing as described above and six rats for each formulation were sacrificed at 0.25, 0.5, 2, 4 and 8 h post-dosing of geniposide-SLNs and geniposide solution. Following complete systemic perfusion with cold saline, tissues including the liver, lung, kidney, heart, brain and spleen were collected, washed with normal saline, blotted dry with filter paper. Ice-cold phosphate buffered saline (0.5 mL) was added to 0.2 g of tissue. Tissues were homogenised in a homogeniser, and stored at 20 C until analysis. Plasma and tissue samples treatment To determine the plasma concentration of geniposide, 200 mL of plasma was added to 100 mL of phosphate buffer (pH 5.0), 50 mL of ascorbic acid (200 mg/mL), then partitioned with 1.0 mL of acetonitrile containing 3.6 mg/mL of hyperoside as the internal standard. After centrifuging at 10 000 g for 15 min, the acetonitrile was evaporated under nitrogen gas to dry and reconstituted with an appropriate volume of mobile phase then 20 mL was subject to HPLC analysis. To determine the tissue concentration of geniposide, 200 mL of tissue (liver, lung, kidney, heart and spleen) homogenates, was added to 100 mL of phosphate buffer (pH 5.0), 50 mL of ascorbic acid (200 mg/mL), then partitioned with 1.0 mL of acetonitrile containing 3.6 mg/mL of hyperoside as the internal standard. The later procedures followed that of plasma samples.
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HPLC analysis
The surface morphology of geniposide-SLNs
The concentration of geniposide in the plasma was determined by HPLC. The HPLC apparatus included a pump (LC-10 A; Shimadzu, Japan), an UV detector (SPD-10 A; Shimadzu, Japan) and a Diamonsil C18 column (5 mm, 4.6 250 mm) was used with a pre-filter (Isolation Technologies) . A mobile phase consisted of acetonitrile and water phase (19:81, v/v) at a flow rate of 1.0 mL/min. The water phase was adjusted to pH 5.0 by phosphoric acid. The detection wavelength was set at 238 nm.
A distribution of sized particles (40 nm) uniformly distributed around the central core was observed. The morphology of geniposide-SLNs was similar as they appear generally well formed and characterised by a spherical shape (Figure 2).
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Analytical method validation To validate the analytical method, the method was validated to demonstrate the specificity, linearity, accuracy and precision of measurements, recovery and stability of samples. The specificity was established by the lack of interference peaks at the retention time for geniposide and the internal standard. The linearity was tested at seven concentration levels covering a range of 0.12–60 mg/mL (0.12, 0.60, 1.20, 2.40, 12, 30 and 60 mg/mL) in plasma and 0.12–30 mg/mL (0.12, 0.48, 0.60, 1.20, 2.40, 12 and 30 mg/mL) in tissue samples. The calibration graphs were plotted by linear regression of the peak area ratios (geniposide to internal standard) against concentrations of geniposide. The accuracy and precision were determined in six replicates of 0.12, 2.40 and 30 mg/mL of geniposide in blank rat plasma and 0.12, 1.20 and 12 mg/mL in blank tissue samples, which were prepared and analysed on 3 consecutive days. The recovery was determined by comparing the peak area obtained from either the extracted plasma or tissue samples with the peak area obtained by the direct injection of the corresponding spiked standard solutions. Three different concentrations of geniposide (0.12, 2.40 and 30 mg/mL in plasma and 0.12, 1.20 and 12 mg/mL in tissue samples) were measured. The stability in rat plasma was examined at room temperature and 20 C. The storage stability at 20 C for 1 month was also evaluated. Triplicate samples of 0.12, 2.40 and 30 mg/mL in plasma and 0.12, 1.20 and 12 mg/mL in tissue samples were determined, respectively.
DSC of geniposide-SLNs DSC curves of geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188 and glyceryl monostearate are given in Figure 3. The DSC curves of poloxamer 188, glyceryl monostearate, geniposide showed melting endotherms at 52.32, 57.07 and 160.73 C. In the case of thermogram of geniposide-SLNs, two peaks were observed at 46.61 C and 51.80 C, which were lower
Data analysis The pharmacokinetic parameters were obtained by compartmental methods using DAS software (Ver. 2.0, Mathematical Pharmacology Professional Committee of China). The area under the plasma concentration–time curve (AUC0–t) was calculated by the trapezoidal rule. The peak plasma concentration (Cmax) and the time to peak concentration (Tmax) were obtained directly from the individual plasma concentration–time profiles. The Student’s t-test was used to analyse differences between both groups. Statistically significant differences were assumed at p50.01.
Results Particle size, zeta potential, EE and DL The geniposide-SLNs formulation showed a particle size 116.5 8.9 nm and PDI 0.283 0.025, both indicating effective and controlled formulation of SLNs using melt-emulsion ultrasonication. The zeta potential of geniposide-SLNs and blank SLN was 10.1 and 11.4 mV, respectively. The EE and DL of geniposide-SLNs were found to be 75.6 1.8% and 10.19%, respectively (Table 1).
Figure 2. Scanning electron micrographs of geniposide loaded SLNs (1 mm and 500 nm).
Table 1. Particle size, total drug content and entrapment efficiency of developed geniposide-SLNs (n ¼ 3). Formulation Geniposide-SLNs
Particle size (nm)
PDI
EE (%)
DL (%)
Zeta potential (mV)
116.5 8.9
0.283 0.025
75.6 1.8
7.56 0.14
10.10 0.36
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Figure 3. Differential scanning calorimetry thermograms of the ingredients and of lipid nanoparticles. (A) Poloxamer 188; (B) glyceryl monostearate; (C) geniposide; (D) lyophilized geniposide-SLNs.
than the peak obtained in the thermogram of poloxamer 188 and glyceryl monostearate, geniposide. In vitro release studies The drug release behaviour in vitro of geniposide-SLNs was investigated using a dialysis membrane in pH 6.8 phosphate buffer (Figure 4). The in vitro geniposide-SLNs release showed triphasic behaviour comprising an initial fast release of free drug, followed by a slow release and finally a delayed release phase starting approximately at 4 h showing almost a controlled release. The mechanism of release was determined by finding the R2 value for each kinetic model Peppas–Korsmeyer, First-order, Higuchi, and Hixon–Crowell model corresponding to the release data obtained. The Peppas–Korsmeyer model was the best (R2 ¼ 0.9916), because the R2 value of the Peppas–Korsmeyer model is much larger than other kinetic models. Method validation and pharmacokinetic studies The method was selective, and there was no interference from endogenous compounds/excipient at the retention time of geniposide. The calibration curve was found to be linear at the tested concentration range for geniposide, i.e. 0.12–60 mg/mL in plasma and 0.12–30 mg/mL in tissue (liver, lung, kidney, brain, heart and spleen) samples. The %RSD and %RE for intra- and inter-day precision and accuracy (n ¼ 6) was below 8.5%. The recoveries of geniposide from plasma, liver, lung, kidney, brain, heart, brain and spleen tissue homogenate were above 86.5%. Geniposide was stable in methanol and in rat plasma at room temperature and at least one month when stored at 20 C. The mean plasma concentration–time profiles for geniposide after single dose intragastric administration of geniposide-SLNs and geniposide solution to rats are shown in Figure 5. The pharmacokinetic parameters of geniposide were calculated using a two-compartmental analysis and the parameters are summarised in Table 2. The geniposide plasma concentrations were higher in rats administered with geniposide-SLNs than those administered with geniposide solution. The Cmax value of geniposide in the geniposide-SLNs was significantly higher than that obtained with geniposide solution. The Tmax in rats treated with geniposide solution was significantly shorter than those treated with
Figure 4. In vitro drug release of geniposide-SLNs and geniposide solution in pH 6.8 phosphate buffer (n ¼ 3).
Figure 5. The mean plasma concentration–time profiles for geniposide after single dose (100 mg/kg) intragastric administration of geniposideSLNs and geniposide solution to rats (n ¼ 6).
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geniposide-SLNs. Compared with the geniposide solution, the t1/2 and MRT were prolonged and the CL and V1/F were increased with geniposide-SLNs. The AUC0–1 values of geniposide administration of geniposide solution and geniposide-SLNs were 353.41 109.43 h mg/L and 6.96 0.82 h mg/L. The latter was 50 times greater than the former. These results showed that the incorporation of geniposide into SLNs resulted in an increased absorption after oral administration. The mean concentration–time profiles of various tissues for the geniposide solution and geniposide-SLNs are presented in Figures 6 and 7. The Cmax in all six tissues except kidney collected tissues appeared at 0.5 h, the rats treated with geniposide solution. In rats treated with geniposide-SLNs, the Cmax appeared at 4 h in the liver, spleen, brain and lung, and at 2 h in the heart and kidney, respectively, and the Tmax was prolonged. The Cmax in each tissue of rats administered with
Table 2. Pharmacokinetic parameters of geniposide after PO administration of geniposide-SLNs (100 mg/kg) and PO administration of geniposide solution (100 mg/kg) to six rats (n ¼ 6).
Parameters Tmax (min) Cmax (mg/mL) t1/2 (h) V1/F (L) CL/F (L/h) AUC0–72 (hmg/ml) AUC0–1 (hmg/ml) MRT (h)
Pure-SLNs (geniposide 100 mg/kg)
Geniposide solution (geniposide 100 mg/kg)
6.08 0.83 6.73 1.28 35.26 5.83 2882.73 731.24 56.59 15.08 270.12 69.23 353.41 109.43 47.60 16.47
0.50 0.13 1.60 0.64 3.28 0.35* 6.79 1.51 5.03 1.25 5.33 0.75 6.96 0.82 7.27 1.36
t1/2, half-life of elimination phase; Tmax, the time of maximum concentration; Cmax, the maximum concentration; AUC0–1, the area under concentration–time curve from time zero to infinity; CL, total plasma clearance; V1, volume of distribution of the central compartment; F, absolute oral bioavailability; data are presented as mean standard deviation (n ¼ 6). *p50.05.
geniposide-SLNs was higher than that administered with geniposide solution. The ratios of Cmax in the liver, spleen, heart, kidney, brain and lung for administered with geniposideSLNs to geniposide solution to rats were 9.47, 2.09, 3.88, 5.70, 3.41 and 6.16, respectively. The AUC0–8 h values after the administration of geniposide solution were in the order of kidney4spleen4liver4heart4lung4brain, but the same figures after administration of geniposide-SLNs were in the order of kidney4liver4heart4lung4spleen4brain. The ratios of AUC0–8 h in the liver, spleen, heart, kidney, brain and lung of the geniposide-SLNs to geniposide solution were 25.93, 4.28, 27.91, 10.15, 5.16 and 16.22, respectively.
Discussion Particle size, zeta potential, EE and DL SLNs were prepared following melt-emulsion ultrasonication and low temperature solidification methods. The particle size determination was performed using Zetasizer NANO ZS which can measure particles in the size of 0.3 nm–10 mm. The particle size is 116.5 nm (below 200 nm), which take advantage of the effect for enhanced drug accumulation. Zeta potential is considered as one of the benchmarks of stability of the colloidal system (Hao et al., 2011). A zeta potential having value 25 mV is considered a stable formulation. However, this rule is not available for the system which contains steric stabilisers. The adsorption of steric stabiliser will decrease the zeta potential because of the shift in the shear plane of the particle (Swathi et al., 2010). In our study, the zeta potential value of geniposide-SLNs was 10.1 mV. The absolute value of zeta potential was lower than reported in the literature, and this might be attributed to poloxamer 188, a non-ionic surfactant which decreases the electrostatic repulsion and stabilises the nanoparticles by forming a coat around their surface (Schwarz et al., 1994). Charge on the nanoparticles has been reported to influence their in vivo behaviour (Mittal et al., 2007). The neutral nanoparticles are expected to remain in circulation over
Figure 6. Quantitative tissue distribution of geniposide in the liver, kidney, brain, spleen, lung and heart after single dose (100 mg/kg) intragastric administration of geniposide-SLNs and geniposide solution to rats (n ¼ 6).
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melting point might be attributed to the change in its crystal lattice after incorporation of geniposide and formulation as nanoparticles (Freitas and Mu¨ller, 1999). It is reported that the increased number of lattice defects in the lipid crystal will cause these changes. Small particle size of SLN could create a high suboptimal state which led to decrease in the melting point of the lipid (Gokce et al., 2008).
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In vitro release studies
Figure 7. The column graphic for comparison of the drug localization according to the AUC of organs after single dose (100 mg/kg) intragastric administration of geniposide-SLNs and geniposide solution to rats (n ¼ 6).
a prolonged period (Albanese et al., 2012). The high EE obtained (75.6%) has never been reported before for other such hydrophilic drugs (Singh et al., 2010; De Pintu Kumar et al., 2011). The high entrapment efficiencies may probably be due to the increased amount of emulsifier and lipid, because increasing the amount of lipid resulted in faster solidification of the nanoparticles. The DL of geniposide-SLNs was found to be 10.19%. The level of drug/lipid ratio and emulsifier has positive influence on drug loading. When the amount of surfactant is increased, the percent of DL is increased, while drug concentration is increased, the amount of excipients increases, which results in reduced drug loading. The surface morphology of geniposide-SLNs The shape and surface morphology of geniposide-SLNs were studied by SEM. For most particles, the morphology is spherical with a smooth surface. The sizes observed under SEM were mostly about 40 nm, and this was not in fair agreement with the determination of particle size using dynamic light scattering (DLS) technique. This may be explained these small particles observed under SEM were placed in terms of a unique arrangement. The size and shape has been demonstrated to influence the uptake of nanoparticles into cells (Rohit and Pal, 2013). It is well known that the smaller the particles the higher they adhere to the tissues surface (Florence, 2004). Particles greater than 200 nm are usually filtered out by the reticular meshwork (Qiu et al., 2010). DSC of geniposide-SLNs Characterization of DSC could penetrate the melting and recrystallization behaviour of SLNs. Figure 3 showed DSC curves of geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188 and glyceryl monostearate without cryoprotectant. The pure geniposide displayed a single sharp endothermic peak at 160.73 C, while the peak of geniposide encapsulated into the SLN showed a shift from 160.73 to 149.61 C, indicating the drug incorporated in SLN was in an amorphous form. The results agree with some already published papers (Lv et al., 2009). The bulk poloxamer 188 and glyceryl monostearate melting point (52.32 and 57.07 C) was detected in the case of thermogram, while a decrease of melting point of poloxamer 188 and glyceryl monostearate (46.61 and 51.80 C) was found. This reduction in
Geniposide-SLNs show an initial phase of very fast release (up to 0.5 h) probably due to the lacking of entrapped geniposide (Figure 4). Geniposide is a water soluble drug, so its incorporation into the outer lipidic layer, and its consequent release into the dialysis bag, may have resulted in a lag phase (about 1 and 2 h). This lag phase is short and free geniposide exhibit a slow phase of release within 6 h. A sustained release was observed post 8 h and suggests homogeneous entrapment of the drug throughout the systems. The initial phase releases 41% of the total geniposide present in the formulation, then other drugs exhibit a slow phase of release and the remaining amount may help to maintain the plasma concentrations for 424 h. Pharmacokinetic studies Various studies have reported that a very low level of geniposide actually gains access into the plasma and tissue concentrations following oral administration due to its high aqueous solubility and poor permeability (Ueno et al., 2001; Tseng and Tsai, 2004; Hou et al., 2008). In our study, we successfully prepared geniposide-SLNs for oral administration, and the AUC0–1 values of geniposide-SLNs were 50 times greater than geniposide solution. Geniposide-SLNs were of a small size (particle size 116.5; Table 1) so that they can be transported significantly across the gastrointestinal system, and their residual time in the gastrointestinal tract will be increased. Increased bioavailability with the use of SLNs had been reported earlier. An ingredient used in the formulation of puerarin SLNs, lectins can increase the adherence of microparticles to the intestinal epithelium and enhance penetration of drugs (Vasir et al., 2003). For example, a 10 times enhancement observed with doxorubicin SLN following i.v. administration (Fundaro` et al., 2000). Following the oral administration of geniposide solution, the mean peak plasma concentration of geniposide was 1.60 0.64 mg/mL at 0.50 0.13 h after oral dosing, whereas, in the case of geniposide-SLNs, a significant higher peak plasma level (6.73 1. 28 mg/mL, p50.01) appeared latter with the Tmax being at 6.08 0.83 h (p50.01, Figure 5). Geniposide-SLNs were of a small size such that they can adhere to the stomach wall, so the peak plasma level is high. Geniposide is a water soluble drug, so its incorporation into the outer lipidic layer, and its consequent release into the gastrointestinal tract, so increase their residual time. In previous study, geniposide shows a small t1/2 of 2–4 h (Long et al., 2011; Sun et al., 2011) indicating its short stay in plasma. In order to maintain the requisite plasma concentration, it may need for repetitive dosing or administering high doses of geniposide daily. This can result in neurotoxicity and hepatotoxicity (Duncan and Barry III, 2004; Tostmann et al., 2007). In our study, the t1/2 of geniposide was 35.26 h which can avoid repetitive dosing or administering high doses. It is essential with high geniposide concentrations in various tissues for its pharmacological roles. Geniposide is usually used for the treatment of the disease of the liver, heart and brain. In our study, the ratios of Cmax in the liver, heart and brain after the administration with geniposide-SLNs to the geniposide solution were 9.47, 3.88 and 3.41, respectively. The ratios of AUC0–8 h in the liver, heart and brain were 25.93, 27.91 and 5.16, respectively.
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It is well known that the increasing concentrations of geniposide in the liver, heart and brain are in favour of its protective effects on hepatic and cardio-cerebrovascular diseases following oral administration of geniposide-SLNs. The blood-brain barrier (BBB) is the main barrier to drug transport into brain, and many drugs are difficult penetration into the brain by BBB under normal conditions. The mechanisms of SLNs penetration into the brain are not completely understood. The ApoE, Apo C-II, albumin and immunoglobulin G seem to be crucial in the sitespecific targeting of the brain (Brioschi et al., 2007). The tissue concentrations of geniposide increased after a single dose oral administration of geniposide-SLNs, especially in its target organs, the liver, spleen, lung and brain, which belong to reticuloendothelial system (RES). The reason of accumulation in RES organs is that the particles smaller than 200 nm are usually expected to apprehend by pass RES and result in prolonged circulation times. These data collectively show that SLNs are a promising delivery system for the enhancement of oral absorption of geniposide.
Conclusions In summary, we prepared geniposide-SLNs using a melt-emulsion ultrasonication method. The characteristics of the preparation were investigated, and geniposide-SLNs with a small particle size and high EE were obtained. Geniposide-SLNs could improve the relative bioavailability more than 50 times, compared to geniposide solution. Furthermore, entrapping geniposide into SLNs could result in improved relative availability of geniposide in the liver, heart and brain. These data collectively support that geniposide-SLNs has the potential to be developed into a product in the future.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported in part by the Department of Education of Shandong Province (Grant Number J11LF31) and the National Nature Science Foundation of China (No. 81102820).
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ORIGINAL ARTICLE
Pharmacokinetics, tissue distribution and relative bioavailability of geniposide-solid lipid nanoparticles following oral administration Fugang Wang, Juan Cao, Jifu Hao, and Ke Liu
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School of Pharmacy, Taishan Medical University, Taian, China
Abstract
Keywords
Geniposide has various pharmacological effects; however, low oral bioavailability limits its clinical utility. This study explores the pharmacokinetics, tissue distribution and relative bioavailability of geniposide-solid lipid nanoparticles (SLNs) following oral administration. The geniposide solution and geniposide-SLNs were orally administered to the rats, respectively. The Cmax value of geniposide in the geniposide-SLNs was significantly higher than that obtained with geniposide solution. Compared with the geniposide solution, the t1/2 and MRT were prolonged; the CL and V1/F were increased with geniposide-SLNs. The AUC0–1values of geniposide-SLNs were 50 times greater than geniposide solution. The ratios of AUC0–8 h in the liver, spleen, heart, kidney, brain and lung of the geniposide-SLNs to geniposide solution were 25.93, 4.28, 27.91, 10.15, 5.16 and 16.22, respectively. Prepared geniposide-SLNs are very helpful for increasing the bioavailability of geniposide. These data suggest that SLNs are a promising delivery system to enhance the oral bioavailability of geniposide.
Bioavailability, geniposide, geniposide-SLNs, pharmacokinetics, tissue distribution
Introduction Geniposide (structure shown in Figure 1) is a major active ingredient in the traditional Chinese medicine, Fructus Gardeniae, which comes from the Gardenia jasminoides ELLIS fruits. Fructus Gardeniae is a popular Chinese herb for the treatment of febrile diseases including jaundice, acute conjunctivitis, epistaxis, hematemesis, hematuria, pyogenic infections and ulcers of the skin Chang and But, 1987). Various beneficial activities of geniposide have been reported, including antitumour (Hsu et al., 1997; Koo et al., 2004), anti-inflammation (Liaw and Chao, 2001; Koo et al., 2006), anti-thrombosis (Suzuki et al., 2001), immunosuppression (Chang et al., 2005), neuroprotection (Lee et al., 2006; Liu et al., 2009), hypoglycemic effect (Wu et al., 2009; Kojima et al., 2011) and promoting cell proliferation (Chen et al., 2010). Although geniposide has various protective effects, owing to the short elimination half-life, it may possibly lead to severe and acute side effects (Somers et al., 2008) because venous administration of frequent and high doses may be needed. But, for the treatment of chronic diseases, oral administration is the most preferred route regarding the conventional drug delivery system. However, the absorption of geniposide in vivo is very poor after oral administration in our previous study, which diminishes its therapeutic effects. Geniposide is soluble in water, but the low permeability making it a potential candidate for delivery via lipidbased nanoparticulate system. As this would not only improve gut permeability, but may also avoid hepatic first pass metabolism of geniposide. This will reduce both the therapeutic dose and side
Address for correspondence: Dr Fugang Wang, School of Pharmacy, Taishan Medical University, Changcheng Road, Taian, Shandong 271016, China. Tel: +86 538 6229751. Fax: +86 538 6229751. E-mail: [email protected]
History Received 12 May 2013 Revised 10 September 2013 Accepted 28 October 2013 Published online 1 April 2014
effects associated with long term use of geniposide. Thus, oral formulation with improved absorption of geniposide is highly desired. To improve the bioavailability of geniposide, we are using the novel drug delivery systems to improve the current drug therapy. Solid lipid nanoparticles (SLNs) are prepared from solid physiological lipids, resulting in a solid particle matrix that can be loaded with variety of drugs (Luo et al., 2011). SLNs can be prepared from biodegradable, fatty acids, triglycerides and phospholipids and so on, which are normal constituents of the human body (Westesen et al., 1997; Rawat et al., 2008; Souto and Mu¨ller, 2010). However, it is a major challenge for encapsulation of hydrophilic materials into the hydrophobic matrix of SLNs (Cortesi et al., 2002). We have found a method to prepare SNPs of hydrophilic drugs with improved entrapment efficiency. In this study, the primary aim is to investigate the pharmacokinetics of geniposide-SLNs after oral administration to the rats in improving the bioavailability of geniposide, and evaluate the tissue distribution (heart, liver, spleen, lung, brain and kidney) of geniposide-SLNs after oral administration to rats.
Materials and methods Materials Geniposide (purity 99%) was purchased from Ci Yuan Biotechnology Co., Ltd (Shanxi, China). Hyperoside (internal standard) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol and acetonitrile were the products of Merck (Darmstadt, Germany). All other chemicals were of analytical grade. SpragueDawley (SD) rats were obtained from Laboratory Animal Center of Shandong University of Tradition Chinese Medicine (Shandong, China). All animal handling and treatments followed the EC Directive 86/609/EEC.
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were the weight of drug added in the system and the weight of lipid added in the system, respectively. The entrapment efficiency (EE) and drug loading (DL) of geniposide-SLNs were calculated according to Equations (1) and (2): W2 W1 100 W2
ð1Þ
W2 W1 100 W2 W1 þ W3
ð2Þ
EEð%Þ ¼
DLð%Þ ¼
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In vitro release studies
Figure 1. Chemical structures of geniposide (MW404).
Preparation of geniposide-SLNs Solid lipid nanoparticles of geniposide were prepared following melt-emulsion ultrasonication method (Arai et al., 2009). The lipidic phase (glyceryl monostearate, geniposide and appropriate ethanol) and the aqueous phase (poloxamer 188 and water) were heated to 10 C above the lipid melt temperature, and the formulation composition had a geniposide/glyceryl monostearate/poloxamer 188 ratio of 0.3/1/1. Then, the organic phase was added into the hot aqueous phase under mechanical agitation. The resulting solution was kept at the same temperature with the same agitation speed. The proportion of two phases formed a microemulsion spontaneously. Hot microemulsion was treated for 5 minutes (work 2 seconds and stand 3 seconds) by a Lab ultrasonic cell pulveriser (JY92-II, Ningbo Scientz Biotechnology Co., Ltd, China) at 600 W, and rapidly cooled by immersing the beaker into ice water (0 C). The prepared geniposide-SLNs were used as such for further studies. Particle size, zeta potential and surface morphology of geniposide-SLNs Particle size and zeta potential were determined by Zetasizer NANO ZS (Malvern, England). To study the morphology like sphericity and aggregation, the sample was examined by Inspect F50 (FEI Co., Ltd, Hillsboro, OR). Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) was performed using a differential scanning calorimeter (DSC-60WS; Shimadzu, Japan). The following samples were placed in aluminium pans under a nitrogen purge for analysis: geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188, glyceryl monostearate. A scan rate of 10 C/minute was used over a temperature range of 35–200 C. Entrapment efficiency and drug loading determination) Geniposide-SLNs was precipitated by adding saturated (NH4)2SO4 solution, centrifugation (CS120GXL, Hitachi, Japan) at 50 000 rpm for 15 minutes, and then collecting the supernatant and solid residue. The solid residue was dispersed in 40 mL phosphate buffered saline (PBS) solution (pH 7.4), and then separated by centrifuge method. Both the drug content in supernatant and that in PBS were regarded as W1. W2 and W3
In vitro release was determined in phosphate buffer (pH 6.8) by dialysis bag method using dialysis membrane with a molecular of 12 000–14 000 Da. Two millilitre of geniposide-SLNs dispersion was placed inside the dialysis bag, tied at both the ends and placed into the preheated dissolution media (40 mL). Stirring was maintained at 100 rpm, a RCZ-8A Drug Dissolution Tester (Tianjin Medical Instrument Factory, China), at 37 0.5 C. Two millilitres aliquots were withdrawn at pre-set time intervals (0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h) and replaced by an equal volume of fresh dissolution medium. After suitable dilution, the samples were analysed by HPLC. Corrected concentration of geniposide was calculated in the test samples using the regression equation of the calibration curve. Drug administration and sampling Healthy Sprague-Dawley rats (weighing 250–300 g) were used and they were fasted overnight with free access to water. Twelve rats were divided into two groups: the geniposide-SLNs group and geniposide solution group. In the former group, animals were administered a single dose of geniposide-SLNs by gastric infusion (equivalent to 100 mg/kg of geniposide). In the latter group, those were administered 100 mg/kg of geniposide suspended in normal saline. Blood samples (0.4 mL) were collected orbital venous at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h after dosing. Heparin was used as an anticoagulant. The blood samples were centrifuged at 10 000 g for 10 minutes to separate the plasma and stored at 20 C until analysis. For the tissue distribution, 60 rats received the same dosing as described above and six rats for each formulation were sacrificed at 0.25, 0.5, 2, 4 and 8 h post-dosing of geniposide-SLNs and geniposide solution. Following complete systemic perfusion with cold saline, tissues including the liver, lung, kidney, heart, brain and spleen were collected, washed with normal saline, blotted dry with filter paper. Ice-cold phosphate buffered saline (0.5 mL) was added to 0.2 g of tissue. Tissues were homogenised in a homogeniser, and stored at 20 C until analysis. Plasma and tissue samples treatment To determine the plasma concentration of geniposide, 200 mL of plasma was added to 100 mL of phosphate buffer (pH 5.0), 50 mL of ascorbic acid (200 mg/mL), then partitioned with 1.0 mL of acetonitrile containing 3.6 mg/mL of hyperoside as the internal standard. After centrifuging at 10 000 g for 15 min, the acetonitrile was evaporated under nitrogen gas to dry and reconstituted with an appropriate volume of mobile phase then 20 mL was subject to HPLC analysis. To determine the tissue concentration of geniposide, 200 mL of tissue (liver, lung, kidney, heart and spleen) homogenates, was added to 100 mL of phosphate buffer (pH 5.0), 50 mL of ascorbic acid (200 mg/mL), then partitioned with 1.0 mL of acetonitrile containing 3.6 mg/mL of hyperoside as the internal standard. The later procedures followed that of plasma samples.
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HPLC analysis
The surface morphology of geniposide-SLNs
The concentration of geniposide in the plasma was determined by HPLC. The HPLC apparatus included a pump (LC-10 A; Shimadzu, Japan), an UV detector (SPD-10 A; Shimadzu, Japan) and a Diamonsil C18 column (5 mm, 4.6 250 mm) was used with a pre-filter (Isolation Technologies) . A mobile phase consisted of acetonitrile and water phase (19:81, v/v) at a flow rate of 1.0 mL/min. The water phase was adjusted to pH 5.0 by phosphoric acid. The detection wavelength was set at 238 nm.
A distribution of sized particles (40 nm) uniformly distributed around the central core was observed. The morphology of geniposide-SLNs was similar as they appear generally well formed and characterised by a spherical shape (Figure 2).
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Analytical method validation To validate the analytical method, the method was validated to demonstrate the specificity, linearity, accuracy and precision of measurements, recovery and stability of samples. The specificity was established by the lack of interference peaks at the retention time for geniposide and the internal standard. The linearity was tested at seven concentration levels covering a range of 0.12–60 mg/mL (0.12, 0.60, 1.20, 2.40, 12, 30 and 60 mg/mL) in plasma and 0.12–30 mg/mL (0.12, 0.48, 0.60, 1.20, 2.40, 12 and 30 mg/mL) in tissue samples. The calibration graphs were plotted by linear regression of the peak area ratios (geniposide to internal standard) against concentrations of geniposide. The accuracy and precision were determined in six replicates of 0.12, 2.40 and 30 mg/mL of geniposide in blank rat plasma and 0.12, 1.20 and 12 mg/mL in blank tissue samples, which were prepared and analysed on 3 consecutive days. The recovery was determined by comparing the peak area obtained from either the extracted plasma or tissue samples with the peak area obtained by the direct injection of the corresponding spiked standard solutions. Three different concentrations of geniposide (0.12, 2.40 and 30 mg/mL in plasma and 0.12, 1.20 and 12 mg/mL in tissue samples) were measured. The stability in rat plasma was examined at room temperature and 20 C. The storage stability at 20 C for 1 month was also evaluated. Triplicate samples of 0.12, 2.40 and 30 mg/mL in plasma and 0.12, 1.20 and 12 mg/mL in tissue samples were determined, respectively.
DSC of geniposide-SLNs DSC curves of geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188 and glyceryl monostearate are given in Figure 3. The DSC curves of poloxamer 188, glyceryl monostearate, geniposide showed melting endotherms at 52.32, 57.07 and 160.73 C. In the case of thermogram of geniposide-SLNs, two peaks were observed at 46.61 C and 51.80 C, which were lower
Data analysis The pharmacokinetic parameters were obtained by compartmental methods using DAS software (Ver. 2.0, Mathematical Pharmacology Professional Committee of China). The area under the plasma concentration–time curve (AUC0–t) was calculated by the trapezoidal rule. The peak plasma concentration (Cmax) and the time to peak concentration (Tmax) were obtained directly from the individual plasma concentration–time profiles. The Student’s t-test was used to analyse differences between both groups. Statistically significant differences were assumed at p50.01.
Results Particle size, zeta potential, EE and DL The geniposide-SLNs formulation showed a particle size 116.5 8.9 nm and PDI 0.283 0.025, both indicating effective and controlled formulation of SLNs using melt-emulsion ultrasonication. The zeta potential of geniposide-SLNs and blank SLN was 10.1 and 11.4 mV, respectively. The EE and DL of geniposide-SLNs were found to be 75.6 1.8% and 10.19%, respectively (Table 1).
Figure 2. Scanning electron micrographs of geniposide loaded SLNs (1 mm and 500 nm).
Table 1. Particle size, total drug content and entrapment efficiency of developed geniposide-SLNs (n ¼ 3). Formulation Geniposide-SLNs
Particle size (nm)
PDI
EE (%)
DL (%)
Zeta potential (mV)
116.5 8.9
0.283 0.025
75.6 1.8
7.56 0.14
10.10 0.36
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Figure 3. Differential scanning calorimetry thermograms of the ingredients and of lipid nanoparticles. (A) Poloxamer 188; (B) glyceryl monostearate; (C) geniposide; (D) lyophilized geniposide-SLNs.
than the peak obtained in the thermogram of poloxamer 188 and glyceryl monostearate, geniposide. In vitro release studies The drug release behaviour in vitro of geniposide-SLNs was investigated using a dialysis membrane in pH 6.8 phosphate buffer (Figure 4). The in vitro geniposide-SLNs release showed triphasic behaviour comprising an initial fast release of free drug, followed by a slow release and finally a delayed release phase starting approximately at 4 h showing almost a controlled release. The mechanism of release was determined by finding the R2 value for each kinetic model Peppas–Korsmeyer, First-order, Higuchi, and Hixon–Crowell model corresponding to the release data obtained. The Peppas–Korsmeyer model was the best (R2 ¼ 0.9916), because the R2 value of the Peppas–Korsmeyer model is much larger than other kinetic models. Method validation and pharmacokinetic studies The method was selective, and there was no interference from endogenous compounds/excipient at the retention time of geniposide. The calibration curve was found to be linear at the tested concentration range for geniposide, i.e. 0.12–60 mg/mL in plasma and 0.12–30 mg/mL in tissue (liver, lung, kidney, brain, heart and spleen) samples. The %RSD and %RE for intra- and inter-day precision and accuracy (n ¼ 6) was below 8.5%. The recoveries of geniposide from plasma, liver, lung, kidney, brain, heart, brain and spleen tissue homogenate were above 86.5%. Geniposide was stable in methanol and in rat plasma at room temperature and at least one month when stored at 20 C. The mean plasma concentration–time profiles for geniposide after single dose intragastric administration of geniposide-SLNs and geniposide solution to rats are shown in Figure 5. The pharmacokinetic parameters of geniposide were calculated using a two-compartmental analysis and the parameters are summarised in Table 2. The geniposide plasma concentrations were higher in rats administered with geniposide-SLNs than those administered with geniposide solution. The Cmax value of geniposide in the geniposide-SLNs was significantly higher than that obtained with geniposide solution. The Tmax in rats treated with geniposide solution was significantly shorter than those treated with
Figure 4. In vitro drug release of geniposide-SLNs and geniposide solution in pH 6.8 phosphate buffer (n ¼ 3).
Figure 5. The mean plasma concentration–time profiles for geniposide after single dose (100 mg/kg) intragastric administration of geniposideSLNs and geniposide solution to rats (n ¼ 6).
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geniposide-SLNs. Compared with the geniposide solution, the t1/2 and MRT were prolonged and the CL and V1/F were increased with geniposide-SLNs. The AUC0–1 values of geniposide administration of geniposide solution and geniposide-SLNs were 353.41 109.43 h mg/L and 6.96 0.82 h mg/L. The latter was 50 times greater than the former. These results showed that the incorporation of geniposide into SLNs resulted in an increased absorption after oral administration. The mean concentration–time profiles of various tissues for the geniposide solution and geniposide-SLNs are presented in Figures 6 and 7. The Cmax in all six tissues except kidney collected tissues appeared at 0.5 h, the rats treated with geniposide solution. In rats treated with geniposide-SLNs, the Cmax appeared at 4 h in the liver, spleen, brain and lung, and at 2 h in the heart and kidney, respectively, and the Tmax was prolonged. The Cmax in each tissue of rats administered with
Table 2. Pharmacokinetic parameters of geniposide after PO administration of geniposide-SLNs (100 mg/kg) and PO administration of geniposide solution (100 mg/kg) to six rats (n ¼ 6).
Parameters Tmax (min) Cmax (mg/mL) t1/2 (h) V1/F (L) CL/F (L/h) AUC0–72 (hmg/ml) AUC0–1 (hmg/ml) MRT (h)
Pure-SLNs (geniposide 100 mg/kg)
Geniposide solution (geniposide 100 mg/kg)
6.08 0.83 6.73 1.28 35.26 5.83 2882.73 731.24 56.59 15.08 270.12 69.23 353.41 109.43 47.60 16.47
0.50 0.13 1.60 0.64 3.28 0.35* 6.79 1.51 5.03 1.25 5.33 0.75 6.96 0.82 7.27 1.36
t1/2, half-life of elimination phase; Tmax, the time of maximum concentration; Cmax, the maximum concentration; AUC0–1, the area under concentration–time curve from time zero to infinity; CL, total plasma clearance; V1, volume of distribution of the central compartment; F, absolute oral bioavailability; data are presented as mean standard deviation (n ¼ 6). *p50.05.
geniposide-SLNs was higher than that administered with geniposide solution. The ratios of Cmax in the liver, spleen, heart, kidney, brain and lung for administered with geniposideSLNs to geniposide solution to rats were 9.47, 2.09, 3.88, 5.70, 3.41 and 6.16, respectively. The AUC0–8 h values after the administration of geniposide solution were in the order of kidney4spleen4liver4heart4lung4brain, but the same figures after administration of geniposide-SLNs were in the order of kidney4liver4heart4lung4spleen4brain. The ratios of AUC0–8 h in the liver, spleen, heart, kidney, brain and lung of the geniposide-SLNs to geniposide solution were 25.93, 4.28, 27.91, 10.15, 5.16 and 16.22, respectively.
Discussion Particle size, zeta potential, EE and DL SLNs were prepared following melt-emulsion ultrasonication and low temperature solidification methods. The particle size determination was performed using Zetasizer NANO ZS which can measure particles in the size of 0.3 nm–10 mm. The particle size is 116.5 nm (below 200 nm), which take advantage of the effect for enhanced drug accumulation. Zeta potential is considered as one of the benchmarks of stability of the colloidal system (Hao et al., 2011). A zeta potential having value 25 mV is considered a stable formulation. However, this rule is not available for the system which contains steric stabilisers. The adsorption of steric stabiliser will decrease the zeta potential because of the shift in the shear plane of the particle (Swathi et al., 2010). In our study, the zeta potential value of geniposide-SLNs was 10.1 mV. The absolute value of zeta potential was lower than reported in the literature, and this might be attributed to poloxamer 188, a non-ionic surfactant which decreases the electrostatic repulsion and stabilises the nanoparticles by forming a coat around their surface (Schwarz et al., 1994). Charge on the nanoparticles has been reported to influence their in vivo behaviour (Mittal et al., 2007). The neutral nanoparticles are expected to remain in circulation over
Figure 6. Quantitative tissue distribution of geniposide in the liver, kidney, brain, spleen, lung and heart after single dose (100 mg/kg) intragastric administration of geniposide-SLNs and geniposide solution to rats (n ¼ 6).
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melting point might be attributed to the change in its crystal lattice after incorporation of geniposide and formulation as nanoparticles (Freitas and Mu¨ller, 1999). It is reported that the increased number of lattice defects in the lipid crystal will cause these changes. Small particle size of SLN could create a high suboptimal state which led to decrease in the melting point of the lipid (Gokce et al., 2008).
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In vitro release studies
Figure 7. The column graphic for comparison of the drug localization according to the AUC of organs after single dose (100 mg/kg) intragastric administration of geniposide-SLNs and geniposide solution to rats (n ¼ 6).
a prolonged period (Albanese et al., 2012). The high EE obtained (75.6%) has never been reported before for other such hydrophilic drugs (Singh et al., 2010; De Pintu Kumar et al., 2011). The high entrapment efficiencies may probably be due to the increased amount of emulsifier and lipid, because increasing the amount of lipid resulted in faster solidification of the nanoparticles. The DL of geniposide-SLNs was found to be 10.19%. The level of drug/lipid ratio and emulsifier has positive influence on drug loading. When the amount of surfactant is increased, the percent of DL is increased, while drug concentration is increased, the amount of excipients increases, which results in reduced drug loading. The surface morphology of geniposide-SLNs The shape and surface morphology of geniposide-SLNs were studied by SEM. For most particles, the morphology is spherical with a smooth surface. The sizes observed under SEM were mostly about 40 nm, and this was not in fair agreement with the determination of particle size using dynamic light scattering (DLS) technique. This may be explained these small particles observed under SEM were placed in terms of a unique arrangement. The size and shape has been demonstrated to influence the uptake of nanoparticles into cells (Rohit and Pal, 2013). It is well known that the smaller the particles the higher they adhere to the tissues surface (Florence, 2004). Particles greater than 200 nm are usually filtered out by the reticular meshwork (Qiu et al., 2010). DSC of geniposide-SLNs Characterization of DSC could penetrate the melting and recrystallization behaviour of SLNs. Figure 3 showed DSC curves of geniposide, lyophilised geniposide-SLNs, poloxamer 188, glyceryl monostearate and mixture of geniposide, poloxamer 188 and glyceryl monostearate without cryoprotectant. The pure geniposide displayed a single sharp endothermic peak at 160.73 C, while the peak of geniposide encapsulated into the SLN showed a shift from 160.73 to 149.61 C, indicating the drug incorporated in SLN was in an amorphous form. The results agree with some already published papers (Lv et al., 2009). The bulk poloxamer 188 and glyceryl monostearate melting point (52.32 and 57.07 C) was detected in the case of thermogram, while a decrease of melting point of poloxamer 188 and glyceryl monostearate (46.61 and 51.80 C) was found. This reduction in
Geniposide-SLNs show an initial phase of very fast release (up to 0.5 h) probably due to the lacking of entrapped geniposide (Figure 4). Geniposide is a water soluble drug, so its incorporation into the outer lipidic layer, and its consequent release into the dialysis bag, may have resulted in a lag phase (about 1 and 2 h). This lag phase is short and free geniposide exhibit a slow phase of release within 6 h. A sustained release was observed post 8 h and suggests homogeneous entrapment of the drug throughout the systems. The initial phase releases 41% of the total geniposide present in the formulation, then other drugs exhibit a slow phase of release and the remaining amount may help to maintain the plasma concentrations for 424 h. Pharmacokinetic studies Various studies have reported that a very low level of geniposide actually gains access into the plasma and tissue concentrations following oral administration due to its high aqueous solubility and poor permeability (Ueno et al., 2001; Tseng and Tsai, 2004; Hou et al., 2008). In our study, we successfully prepared geniposide-SLNs for oral administration, and the AUC0–1 values of geniposide-SLNs were 50 times greater than geniposide solution. Geniposide-SLNs were of a small size (particle size 116.5; Table 1) so that they can be transported significantly across the gastrointestinal system, and their residual time in the gastrointestinal tract will be increased. Increased bioavailability with the use of SLNs had been reported earlier. An ingredient used in the formulation of puerarin SLNs, lectins can increase the adherence of microparticles to the intestinal epithelium and enhance penetration of drugs (Vasir et al., 2003). For example, a 10 times enhancement observed with doxorubicin SLN following i.v. administration (Fundaro` et al., 2000). Following the oral administration of geniposide solution, the mean peak plasma concentration of geniposide was 1.60 0.64 mg/mL at 0.50 0.13 h after oral dosing, whereas, in the case of geniposide-SLNs, a significant higher peak plasma level (6.73 1. 28 mg/mL, p50.01) appeared latter with the Tmax being at 6.08 0.83 h (p50.01, Figure 5). Geniposide-SLNs were of a small size such that they can adhere to the stomach wall, so the peak plasma level is high. Geniposide is a water soluble drug, so its incorporation into the outer lipidic layer, and its consequent release into the gastrointestinal tract, so increase their residual time. In previous study, geniposide shows a small t1/2 of 2–4 h (Long et al., 2011; Sun et al., 2011) indicating its short stay in plasma. In order to maintain the requisite plasma concentration, it may need for repetitive dosing or administering high doses of geniposide daily. This can result in neurotoxicity and hepatotoxicity (Duncan and Barry III, 2004; Tostmann et al., 2007). In our study, the t1/2 of geniposide was 35.26 h which can avoid repetitive dosing or administering high doses. It is essential with high geniposide concentrations in various tissues for its pharmacological roles. Geniposide is usually used for the treatment of the disease of the liver, heart and brain. In our study, the ratios of Cmax in the liver, heart and brain after the administration with geniposide-SLNs to the geniposide solution were 9.47, 3.88 and 3.41, respectively. The ratios of AUC0–8 h in the liver, heart and brain were 25.93, 27.91 and 5.16, respectively.
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It is well known that the increasing concentrations of geniposide in the liver, heart and brain are in favour of its protective effects on hepatic and cardio-cerebrovascular diseases following oral administration of geniposide-SLNs. The blood-brain barrier (BBB) is the main barrier to drug transport into brain, and many drugs are difficult penetration into the brain by BBB under normal conditions. The mechanisms of SLNs penetration into the brain are not completely understood. The ApoE, Apo C-II, albumin and immunoglobulin G seem to be crucial in the sitespecific targeting of the brain (Brioschi et al., 2007). The tissue concentrations of geniposide increased after a single dose oral administration of geniposide-SLNs, especially in its target organs, the liver, spleen, lung and brain, which belong to reticuloendothelial system (RES). The reason of accumulation in RES organs is that the particles smaller than 200 nm are usually expected to apprehend by pass RES and result in prolonged circulation times. These data collectively show that SLNs are a promising delivery system for the enhancement of oral absorption of geniposide.
Conclusions In summary, we prepared geniposide-SLNs using a melt-emulsion ultrasonication method. The characteristics of the preparation were investigated, and geniposide-SLNs with a small particle size and high EE were obtained. Geniposide-SLNs could improve the relative bioavailability more than 50 times, compared to geniposide solution. Furthermore, entrapping geniposide into SLNs could result in improved relative availability of geniposide in the liver, heart and brain. These data collectively support that geniposide-SLNs has the potential to be developed into a product in the future.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported in part by the Department of Education of Shandong Province (Grant Number J11LF31) and the National Nature Science Foundation of China (No. 81102820).
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