- Email: [email protected]
Nanostructured lipid carriers versus microemulsions for delivery of the poorly water-soluble drug luteolin
International Journal of Pharmaceutics 476 (2014) 169–177
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Nanostructured lipid carriers versus microemulsions for delivery of the poorly water-soluble drug luteolin Ying Liu 1, Lan Wang 1, Yiqing Zhao, Man He, Xin Zhang, Mengmeng Niu, Nianping Feng * School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2014 Received in revised form 12 September 2014 Accepted 28 September 2014 Available online 1 October 2014
Objective: Nanostructured lipid carriers and microemulsions effectively deliver poorly water-soluble drugs. However, few studies have investigated their ability and difference in improving drug bioavailability, especially the factors contributed to the difference. Thus, this study was aimed at investigating their efficiency in bioavailability enhancement based on studying two key processes that occur in NLC and ME during traverse along the intestinal tract: the solubilization process and the intestinal permeability process. Methods: The nanostructured lipid carriers and microemulsions had the same composition except that the former were prepared with solid lipids and the latter with liquid lipids; both were evaluated for particle size and zeta potential. Transmission electron microscopy, differential scanning calorimetry, and X-ray diffraction were performed to characterize their properties. Furthermore, in vitro drug release, in situ intestinal absorption, and in vitro lipolysis were studied. The bioavailability of luteolin delivered using nanostructured lipid carriers in rats was compared with that delivered using microemulsions and suspensions. Results: The in vitro analysis revealed different release mechanisms for luteolin in nanostructured lipid carriers and microemulsions, although the in situ intestinal absorption was similar. The in vitro lipolysis data indicated that digestion speed and extent were higher for microemulsions than for nanostructured lipid carriers, and that more of the former partitioned to the aqueous phase. The in vivo bioavailability analysis in rats indicated that the oral absorption and bioavailability of luteolin delivered using nanostructured lipid carriers and microemulsions were higher than those of luteolin suspensions. Conclusion: Nanostructured lipid carriers and microemulsions improved luteolin’s oral bioavailability in rats. The rapid lipid digestion and much more drug solubilized available for absorption in microemulsions may contribute to better absorption and higher bioavailability. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Nanostructured lipids carriers Microemulsions Luteolin In situ intestinal absorption In vitro lipolysis Oral bioavailability
1. Introduction Luteolin (Fig. 1) is a natural 4-dihydroxy flavone compound isolated from Reseda odorata. It has antioxidative, anti-inflammatory/allergic (Bor et al., 2006; Chen et al., 2008; Hougee et al., 2005; Günter et al., 2008; Kayoko et al., 2001), antibacterial (Lv et al., 2009), and anticancer (Koen et al., 2005; Leunga et al., 2006) properties, and has been shown to inhibit protein kinase C
* Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, China. Tel.: +86 21 5132 2198; fax: +86 21 5132 2198. E-mail addresses: [email protected], [email protected] (N. Feng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2014.09.052 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
(Leunga et al., 1989). In clinical application, luteolin is mainly used to treat chronic bronchitis and other respiratory diseases. However, it has very low bioavailability after oral administration because of its poor solubility in aqueous media (Sanghoo et al., 2003). Therefore, there is a clear need to improve the bioavailability of luteolin to increase its potential in clinical application. The most popular approach for improving the oral bioavailability for poorly water-soluble drugs is the use of lipid-based formulations (Pouton, 2000; Humberstone and Charman, 1997; Constantinides, 1995). Lipid vehicles have been used to enhance the absorption of lipophilic drugs for many years. Their mechanisms of action include enhanced dissolution and solubilization of the co-administered lipophilic drug by stimulation of biliary and pancreatic secretions, prolongation of gastrointestinal tract
170
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
OH
OH
O OH
O
OH
Fig. 1. Chemical structure of luteolin.
residence time, stimulation of lymphatic transport, increased intestinal wall permeability, and reduced metabolism and efflux activity (Dahan et al., 2006; Cershanik and Benita, 2000). In the early 1990s, solid lipid nanoparticles (SLN) were developed as an alternative colloidal lipid system for controlled drug delivery. Nanostructured lipid carriers (NLC) composed of a solid lipid matrix with some amount of liquid lipids are new-generation SLN (Muller et al., 2002), which can overcome the limitations of SLN. The presence of liquid lipid creates more imperfections in the matrix to allow accommodation of more drug molecules compared to those in SLN. Furthermore, drug loading is enhanced, drug expulsion during storage is minimized, and the drug release profile can be modulated by varying the lipid matrix (Radtke et al., 2005). Microemulsions, another popular lipid-based delivery approach, are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules. An increasing number of reports suggest that lipid-based microemulsions can be used to enhance the oral bioavailability of drugs, including peptides (Lu et al., 2013; Sane et al., 2013; Wu et al., 2006; Lu et al., 2012). Drug delivery advantages of microemulsions include improved drug solubilization and protection against enzymatic hydrolysis, as well as the potential for enhanced absorption due to surfactant-induced membrane fluidity and permeability changes. There are some research reports on the ability of NLC and microemulsions to enhance the oral bioavailability of poorly water-soluble drugs (Shangguan et al., 2014; Tian et al., 2013; Qi et al., 2012). However, the extent between the two formulations in bioavailability enhancement has not been fully established yet, especially what factors contribute to the difference. After NLC or ME is located within the GI tract, the fate of the loaded drugs is of first essential for efficient absorption, which are determined by the dispersion process and subsequent digestion process (Devraj et al., 2013; Porter et al., 2007). The incorporated drugs are likely to be solubilized within various micelle or micelle similar vehicles, which are formed by the product of lipid digestion to the lipid carriers together with endogenous ingredients such as bile salts and phospholipids. On the other hand, the co-administered drugs have the tendency of precipitation upon loosing the protection of hydrophilic formulation components due to the change of aqueous surroundings during dispersion or lipid digestion process
(Bakala N’Goma et al., 2012; Anby et al., 2012; Thomas et al., 2012). Due to lipid components and the surfactants containing ester groups from NLC and ME are all the substrates of the hydrolysis reaction by pancreatic lipase, it is rational to deduce that lipid digestion process is inevitable in vivo, which may influence drug absorption and bioavailability. Thus, the aim of present study was to evaluate their efficiency in bioavailability enhancement based on the investigation of two key processes that occur in NLC and ME during traverse along the intestinal tract: the solubilization process and the intestinal permeability process. A dynamic in vitro lipolysis assay was used to determine drug solubilization, and intestinal permeability was tested with an in situ rat intestinal absorption model. The results of in vitro and in situ analyses were subsequently compared with in vivo oral bioavailability data. 2. Materials and methods 2.1. Materials Luteolin was obtained from Jiangxi Bencaotiangong Medical Technology Co., Ltd., China. Glyceryl palmitostearate (Precirol ATO1 5) and caprylocaproyl macrogol-8 glycerides (Labrasol1) were donated by Gattefossè, France. Polyoxyl castor oil (Cremophor ELP) was donated by BASF, Germany. Tris maleate, sodium taurodeoxycholate (NaTDC), and porcine pancreatin were purchased from Sigma–Aldrich, USA. 4-Bromophenylboronic acid (4-BPB) was purchased from Acros Organics, Belgium. Phosphatidylcholine (PC) was purchased from Lipoid, Germany. Methanol (HPLC grade) was purchased from Merck, Germany. Phosphoric acid, perchlorate, ethyl acetate, glucose, mannose, sucrose, lactose, and mycose were purchased from Shanghai Chemical Reagent Co., China. All other chemicals were HPLC or analytical grade. 2.2. Preparation of luteolin-NLC (LT-NLC) and luteolin-ME (LT-ME) The composition of the two preparations is shown in Table 1. Hot high-pressure homogenization (HPH) (GEA Niro Soavi NS1001L, Homogenizer systems, Italy) was used to prepare LT-NLC. Briefly, solid lipid, liquid lipid, and surfactant were heated at 70 C with continuous stirring to homogeneously mix the lipids. The drug was dissolved in the melted mixture. Then, distilled water heated to 70 C was poured into the mixture and homogeneously dispersed using a homogenizer (IKA1 RCT, Germany) at 10,000 rpm for 10 min, while maintaining the temperature at 70 C. This pre-emulsion was further homogenized for five cycles at 800 bar. The dispersion was immediately placed in an ice bath with gentle stirring for 10 min before further use. LT-ME was prepared by dissolving luteolin in a mixture of Labrasol and Cremophor ELP. Then, distilled water was dropped into the mixture with gentle stirring until uniform and transparent preparations were formed. 2.3. Determination of particle size, polydispersity index (PDI), and zeta potential Particle size (z-average diameter) and PDI were measured with a dynamic light scattering technique using a Malvern Zetasizer
Table 1 Formulation composition of LT-NLC and LT-ME. Formulation
Ingredient (%, w/w) Luteolin
Precirol ATO 5
Labrasol
Cremophor ELP
Distilled water
LT-NLC LT-ME
0.01 0.01
3.48 –
0.75 4.23
2.61 2.61
93.15 93.15
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Table 2 Particle size, PDI, and zeta potential of LT-NLC and LT-ME.
LT-NLC LT-ME
Particle size/nm
PDI
Zeta potential/mv
43.89 4.52 12.98 0.14
0.219 0.007 0.202 0.038
19.42 0.79 4.39 0.32
(n = 3).
Nano ZS90 (Malvern Instruments, UK) at 25 C and an angle of 90 . Zeta potential was measured with the same instrument. Before measurement, the preparations were diluted with deionized water to achieve a suitable scattering intensity. Each sample was analyzed in triplicate. 2.4. Characterization of LT-NLC 2.4.1. Determination of drug loading and encapsulation efficiency Drug loading and encapsulation efficiency were determined using a previously reported method (Lu et al., 2009). Briefly, after
171
filtration through a 0.45-mm filter (Sartorius, Germany), the filtrate was diluted 100-fold with acetone. It was mixed well using a cyclomixer to completely extract the drug from lipid to acetone. Then, the drug content was analyzed by HPLC. The amount of soluble unencapsulated drug was measured by ultrafiltration using centrifugal filter tubes with a molecular weight cut-off of 300 kDa (Millipore, Ireland). Briefly, a 200-mL aliquot of LT-NLC was placed into the centrifugal filter tube and centrifuged at 5827 g for 10 min. The amount of drug in the aqueous phase was determined by HPLC. Drug loading and encapsulation efficiency were calculated using Eqs. (1) and (2), respectively. Load contentð%Þ ¼
Ws 100% W lipid
Entrapment efficiencyð%Þ ¼
Ws 100% W total
(1)
(2)
where Ws represents the amount of luteolin in the NLC, Wlipid is the weight of the vehicle, and Wtotal represents the amount of luteolin used in the formulation. The HPLC assay was performed as follows. A reverse phase C18 column (Diamond C18, 250 mm 4.6 mm, 5 mm; Dikma) was used. A mixture of methanol (40%) and 0.1% aqueous solution of phosphoric acid (60%) was used as the mobile phase. Column temperature, mobile phase flow rate, injection volume, and detection wavelength were set at 30 C, 1 mL/min, 20 mL, and 348 nm, respectively. The assay method was validated and the calibration curve was linear (r = 0.9999) within the 0.60–39.96 mg/ mL range. 2.4.2. Transmission electron microscopy (TEM) The morphology of NLC was determined by TEM (JEM-1230, JEOL, Japan). The samples were diluted with distilled water, and then stained with 1% (w/v) phosphotungstic acid and placed on copper grids with films. After air-drying for 3 h at room temperature, the specimens were analyzed by TEM. 2.4.3. Differential scanning calorimetry (DSC) analysis NLC dispersions were lyophilized (Alpha 1-4, Marein Christ, Germany) prior to DSC analysis. Solid lipid (Precirol ATO 5), drug (luteolin), physical lipid/drug mixture, cryoprotectant mixture (mannose, mycose), and drug-loaded NLC were subjected to DSC (DSC 204 F1 Phoenix1, Netzsch, Germany) analysis. Briefly, samples were kept in standard aluminum pans and sealed. DSC analysis was performed at 10 C/min at temperatures of 30 to 350 C in a nitrogen environment. An empty, sealed pan was used as a reference. The thermograms were recorded.
Fig. 2. Transmission electron micrograph of (A) blank-NLC and (B) LT-NLC.
Fig. 3. DSC curve of (A)LT-NLC; (B) physical mixture; (C) mixture of cyroprotectant; (D) Precirol ATO 5; (E) luteolin.
172
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
Q ½1 Ph:redout =Ph:redin L
2.4.4. X-ray diffraction (XRD) analysis XRD analysis was conducted using a powder X-ray diffractometer (D/MAX 2550 VB/PC, Rigaku, Japan), and Cu/Ka radiation was used as the X-ray source. Samples were placed in the glass holders and scanned from 3 to 50 with a scan angular speed (2u/min) of 8 /min at an operating voltage of 40 kV and a current of 100 mA.
NWF ¼
2.5. In vitro release
Ph. redout and Ph. redin are the outlet and inlet concentrations of the non-absorbable water flux marker phenol red, Cin is the inlet concentration, and Cout is the outlet concentration of compound, which is corrected for volume change in the segment by using the phenol red concentration in the outlet and inlet tubing. Q is the flow rate (0.2 mL/min), L is the length of the segment, and r is the rat intestinal radius.
In vitro release studies were carried out (Liu et al., 2011) in 100 mL of a release medium composed of PBS (pH 7.4) containing 0.5% Tween 80 at 37 C. Preparations (4 mL) were added to a pretreated dialysis bag, then they were immersed in a shaker water bath with shaking speed of 100 strokes/min. One milliliter of the release medium was removed at predetermined time intervals, and the volume was replaced by adding 1 mL of fresh medium. Luteolin concentration in the removed release medium was measured by HPLC. 2.6. In situ intestinal absorption study The experiment was approved by the institutional animal care and use committee, Shanghai University of T.C.M. Adult male Sprague-Dawley rats (weight, 300 20 g) were obtained from the shanghai laboratory animal center. They were housed in air-conditioned quarters with a 12-h light/12-h dark photoperiod schedule. The animals received a standard laboratory diet and tap water ad libitum. The rats were randomly divided into two groups (NLC and ME). In each group, three concentrations of luteolin (2, 5, and 10 mg/mL) were used in separate experiments. The anesthetized rats were restrained in a supine position on a board with temperature maintained at 37 C. A small midline incision was made in the abdomen and a 10-cm section of the small intestine was cannulated. Blank perfusion buffer was infused for 10 min by using a syringe pump followed by perfusion with different concentrations (2, 5, and 10 mg/mL) of luteolin at a flow rate of 0.2 mL/min for 90 min. Outlet samples were collected in microtubes at appropriate intervals (30, 40, 50, 60, 70, and 80 min). The sample volume for each time interval was 2 mL. The length and the radius of segment were measured when the experiment was completed. Samples were stored at 20 C until analysis. Samples from the perfusion study were filtered and directly injected into the HPLC column and required no further preparation prior to analysis. Effective permeability coefficients (Peff) were calculated from the steady-state concentrations of compounds in the collected perfusate, which were considered to be reached when the concentration of phenol red was at a steady state. Concentrations reached a steady state about 40 min after the start of perfusion, which was confirmed by plotting the ratio of the outlet to inlet concentration (corrected for water transport) versus time. The intestinal net water flux (NWF, mL/h/cm) and Peff were calculated using Eqs. (3) and (4), respectively.
Fig. 4. X-ray-diffraction patterns of (A) physical mixture; (B) cryoprotectant; (C) Precirol ATO 5; (D) LT-NLC; (E) blank-NLC; (F) luteolin.
Peff ¼
Q lnðC in =C out Þ 2p rL
(3)
(4)
2.7. In vitro lipolysis study The dynamic in vitro lipolysis experiment was designed to achieve pseudo-physiological conditions. The in vitro lipolysis study was carried out as previously reported (Dahan and Hoffman, 2006). The experimental medium, comprising 29.25 mL of digestion buffer (50 mM Tris maleate, 150 mM NaCl, and 5 mM CaCl2, pH 7.5) containing 5 mM taurocholic acid and 1.25 mM PC (conditions mimicking fasted state GIT), was continuously stirred at 100 rpm and maintained at 37 C. Then, 6.75 mL of the test formulation (NLC or ME, stirred and gently heated to 37 C, and freshly prepared 30 min before each experiment) was dispersed in the medium and stirred for 15 min. Fresh pancreatin extract was prepared by adding 1 g of porcine pancreatin powder to 5 mL of digestion buffer, stirring for 15 min, and centrifuging as previously described (Sek et al., 2001). Approximately 4 mL of the pancreatin extract (containing 40,000 tributyrin units of pancreatic lipase activity) was added to the medium to initiate the enzymatic digestion of the formulation. A pH titrator unit (TitraLab 856 pH-Stat, Radiometer, France) was used to maintain the pH at 7.5 throughout the experiment. The experiment was continued for 60 min, in which time the enzymatic digestion process was completed, as indicated by the completion of the pH titration. The intestinal absorption of luteolin after lipolysis was also determined. Medium from the in vitro lipolysis experiment, collected immediately after the completion of the lipolysis process, was added to the perfusion buffer to start the in situ intestinal absorption experiment. 2.8. In vivo bioavailability The animal experiments were approved by the institutional animal ethical committee of Shanghai University of TCM. Male Sprague-Dawley rats (weight, 300 20 g) were randomly divided into 3 groups of 5 animals each. Prior to drug administration, the
Fig. 5. In vitro release curves of luteolin from LT-NLC and LT-ME.
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Table 3 Permeability coefficients (Peff) for LT-NLC and LT-ME determined in rats (n = 3). Group
Cin (mg/mL)
Mean Peff (103 cm/min)
Mean NWF (mL/min/cm)
LT-NLC
2 5 10 2 5 10
2.57 0.97 4.78 2.74 5.10 3.04 2.38 1.61 6.36 2.50 12.70 4.79
0.00304 0.000787 0.00039 0.001146 0.00062 0.001951 0.00057 0.000458 0.00259 0.001019 0.00014 0.003642
LT-ME
animals were fasted for 12 h with free access to water. The test preparations were LT-NLC, LT-ME, and LT-suspensions. LT-ME and LT-NLC were prepared as described in Section 2.2. LT-suspensions were prepared by dispersing luteolin in sodium carboxymethyl cellulose (CMC-Na) solution containing glycerin. The rats were administered 3.46 mg/kg luteolin with each preparation by oral gavage. Blood samples (1 mL) were collected into heparinized tubes by retro-orbital puncture 0, 0.017, 0.25, 0.5, 1, 2, 3, 5, 8, 12, 24, and 48 h after treatment. Blood samples were centrifuged at 8000 rpm for 5 min. Plasma was kept at 20 C until analysis as described below. Two-hundred microliters of 6.0% perchloric acid was added slowly to 200 mL of plasma to precipitate protein completely. Luteolin was extracted with 3.0 mL of ethyl acetate by vortexing for 5 min. After centrifugation at 8000 rpm for 10 min, 2.40 mL of the organic phase supernatant was carefully transferred to another tube and nitrogen flow-dried. The residue was reconstituted in 100 mL of the mobile phase; the mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was injected into the HPLC system. HPLC analysis was performed on a Diamond C18 column (250 mm 4.6 mm, 5 mm). Optimum separation was carried out with a mobile phase composed of methanol and 0.1% phosphoric acid aqueous solution (55:45, v/v) at a flow rate of 1.0 mL/min. The injected volume of sample was 30 mL, and the detection wavelength and column temperature were set at 348 nm and 30 C, respectively.
173
3. Results and discussion 3.1. Particle size, PDI, and zeta potential The mean particle size of LT-NLC and LT-ME is shown in Table 2. Both preparations had a particle size of less than 50 nm with a low PDI. The ME had a smaller size of 12.98 nm than the NLC (43.89 nm), which may be due to the formation composition difference in oil phase. In the case of NLC, Precirol ATO 5 (glyceryl palmitostearate) was used as solid lipid of NLC, and formed its core. The liquid lipid of labrasol was reported to be well entrapped within solid lipid matrix of Precirol ATO-5 (Doktorovová et al., 2010). In addition, the liquid lipid would be located near or at the surface of lipid nanoparticles, as suggested by Jores et al. (2005) which attributes to physical characteristics such as particle size and zeta potential. While the lipid of ME was totally composed by Labrasol (PEG-6 caprybic/capric triglycerides). Labrasol, except application as lipid of nanocarrier, it is often used as solubilizer and wetting agents, which has relative stronger ability to be dispersed in aqueous surroundings, and therefore attributed to smaller particle size of ME. 3.2. Characterization of LT-NLC
Pharmacokinetic parameters were determined using DAS 2.1.1 software. Statistical analysis for the pharmacokinetic parameters was carried out using one-way analysis of variance (ANOVA). A p value of <0.05 was considered statistically significant.
The drug loading and encapsulation efficiency of the LT-NLC were 0.15% and 95.63%, respectively. The morphology of NLC determined by TEM is shown in Fig. 2. The particles had almost spherical and uniform shapes and were well dispersed. DSC was used to investigate the drug state and crystallization in the compounds and NLC by determining the variations of temperature and energy at phase transition. Fig. 3 shows the DSC curves of Precirol ATO 5, luteolin, cryoprotectant mixture, their physical mixture, and drug-loaded NLC lyophilized powder. The thermogram of luteolin showed a melting peak at 307.5 C. The melting peak for Precirol ATO 5 was observed at 60.4 C, and those for the cryoprotectant mixture at 101.0 and 168.5 C. The melting peaks of luteolin were distinct, whereas those of Precirol ATO 5 and the cryoprotectant mixture had almost same value in the curve as the physical mixture. During the heating process, luteolin was dissolved in the melted solid lipid, which might have led to the disappearance of the peak. With the exception of Precirol ATO 5, all endothermic peaks disappeared in LT-NLC powder, and a new peak appeared at 152.9 C, which suggested that luteolin in the NLC was in an amorphous rather than a crystalline state, to form a solid solution within the matrix of nanoparticles.
Fig. 6. In vitro lipolysis rate of LT-NLC and LT-ME.
Fig. 7. Partition of luteolin in aqueous phase after in vitro lipolysis.
2.9. Data analysis
174
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
effect of drug diffusion was predominant and a smaller amount of drug was retained in the core of the NLC than in the shell. The release profile of LT-ME was fitted to the Higuchi model and the equation was as follows: F = 1.214 t1/2 (r = 0.9665). The results indicated that the release mechanisms of LT-NLC and LT-ME were different. 3.4. In situ intestinal absorption study
Fig. 8. Partition of luteolin in the pellet phase after in vitro lipolysis.
XRD was used to identify the physical state of luteolin incorporated in NLC. The patterns of solid lipid (Precirol ATO 5), drug (luteolin), physical mixture (lipid, drug, and cryoprotectant mixture), cryoprotectant mixture (mannose, mycose), drug-loaded NLC, and drug-free NLC are shown in Fig. 4. For pure luteolin, significant diffraction peaks, indicating that luteolin was in the crystalline form, were detected at 2u scattered angles of 6.879 , 13.561, 13.840 , 14.540 , and 16.280 . The solid–lipid (Precirol ATO 5) pattern showed two characteristic wide peaks (5.260 and 21.460 ), indicating that the bulk matrix (Precirol ATO 5) was also in the crystalline form. There was no distinct peak for LT-NLC, suggesting that it was not crystalized. Some characteristic peaks of Precirol ATO 5 in NLC disappeared and the position of the 21.460 peak shifted forward and its intensity decreased. The results suggest that luteolin was in the amorphous, rather than crystalline state in NLC, and the bulk matrix (Precirol ATO 5) was in a lesserorder crystal. 3.3. In vitro release We used the dialysis bag diffusion technique to investigate luteolin release from LT-NLC and LT-ME in PBS (pH 7.4). The mean cumulative percent release versus time curves are shown in Fig. 5. Compared to the release of LT-ME, the cumulative percent release of LT-NLC in PBS at 0.5 h was approximately 14%. LT-NLC showed an initial burst release, suggesting that there was unencapsulated drug attached to the surface of the particles. A sustained release profile was observed for both LT-NLC and LT-ME, which could increase the circulation time of luteolin, leading to prolonged drug residence time in the systemic circulation and better bioavailability. The release profile of the drug from NLC in PBS was fitted to the Ritger-Peppa’s kinetics model and the equation was as follows: F = 2.369 t0.433 (r = 0.9859). Based on the model, the value of n was 0.433 (0.43 < n < 1.0), suggesting that luteolin release from NLC was because of drug diffusion and lipid matrix erosion. The n value was close to 0.45, indicating that the
The drug amount and total volume of perfusate changed continuously during the experiment because of the absorption in the GI tract. Therefore, to determine the volume change of the perfusate in the intestinal perfusion experiment, phenol red was added into the perfusate solution as a non-absorbed marker. The mean Peff values for different concentrations of LT-NLC and LT-ME as well as the mean net water fluxes, determined with the single pass intestinal perfusion technique are listed in Table 3. The Peff values for different concentrations of LT-NLC were not statistically different (p = 0.433). However, there were differences between the Peff values for different concentrations of LT-ME (p = 0.022). This may be because of the presence of the surfactant in LT-ME. The compositions of LT-ME and LT-NLC developed in this study were similar, except that the former contained liquid and the latter solid lipid. The liquid lipid used was Labrasol, a caprylocaproyl macrogolglyceride, which had a higher HLB value (14), indicating that it had surfactant properties, which could affect permeability. However, further investigation is necessary to elucidate the exact mechanism. There were no differences between the following concentrations of LT-NLC and LT-ME: 2 mg/mL (p = 0.868), 5 mg/mL (p = 0.503), and 10 mg/mL (p = 0.081). 3.5. In vitro lipolysis study During the process of in vitro lipolysis of lipid-based formulations, free fatty acids are formed as a result of lipid degradation. The release of the fatty acids caused a decrease in pH, and NaOH (0.2 M) was added to keep the pH value constant. As a result, NaOH decrease was used to estimate the degradation velocity of the lipid by pancreatic lipase (Fig. 6). In the first 5 min, there was a high initial decrease of NaOH, corresponding to a high output of free fatty acids. The titration curves indicated that the digestion speed was higher for LT-ME than for LT-NLC in the first 20 min, and subsequently LT-ME degradation was slower than that of LT-NLC. This result may be because of the solid lipid present in NLC. Previous studies have shown that solid lipids are still digested by lipase, but the digestion rate is lower than that of liquid lipids (Olbrich et al., 2002). In this study, the existence of solid lipid in NLC may lead to a slower digestion rate compared to that of ME.
Table 4 T-test for LT-NLC and LT-ME Peff values after digestion.
LT-NLC LT-ME F P
Mean Peff (103 cm/min)
Mean NWF (mL/min/cm)
54.36 4.97 64.55 6.55 4.615 0.098
0.00247 0.000979 0.002723 0.00198 – –
Fig. 9. Plasma concentration profile of luteolin after oral administration of LT-NLC, LT-ME and LT-suspensions in rats (n = 5).
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
175
Table 5 Pharmacokinetic parameters of luteolin after oral administration of LT-NLC, LT-ME, and LT-suspensions in rats (n = 5).
LT-NLC LT-ME LT-suspension
AUC0–t (mg h/L)
AUC0–1(mg h/L)
MRT0–t (h)
T1/2z (h)
Tmax (h)
CLz/F (L/h/kg)
3516.552 6045.463 682.75
10663.64 16222.6 1152.713
11.614 4.539 2.367
9.912 4.501 1.238
2.6 4.6 0.25
0.44 0.373 4.404
During lipolysis, samples were collected at specific time points and the lipase activity was inhibited immediately with 4-bromobenzeneboronic acid. Each sample was ultracentrifuged and three phases were formed: (1) the oil phase, (2) the aqueous phase, and (3) the pellet. The aqueous phase is of particular interest for drug solubilization in the GI tract, because drug absorption is believed to happen during this phase (Mu et al., 2013). The results of luteolin partitioning in the aqueous phase after in vitro lipolysis of LT-NLC and LT-ME are shown in Fig. 7. The percentage of Luteolin participated in the aqueous phase increased gradually for both NLC and ME. The product of lipid digestion with bile salts and phospholipids form micelles, or mixed micelles, or other lipid vehicles, in which drug may be incorporated into these colloidal systems, and thus facilitate drug solubilization. In the first 30 min, drug content in the aqueous phase was higher for LT-NLC than for LT-ME, while after 45 min, the pattern reversed. In the case of NLC, part of Luteolin may be absorbed or locate at the surface of NLC, burst release occurred upon NLC dispersion into aqueous media as shown in Fig. 5. This burst release phenomenon may contribute to the higher drug content in the aqueous phase for LT-NLC than for LT-ME in the first 30 min. Labrasol was used as a liquid oil in NLC and ME formulation. Due to the lipid hydrolysis by pancreatic lipase, the lipolytic products together with other components such as bile salts and phospholipids may form micelles, which may increase the solubilized drug in the system. In addition, the properties of dispersion media such as CMC, surface tension, diffusion capacity have been shown to influence drug dissolve in aqueous media (Paula et al., 2011). Labrasol as a pharmaceutical excipient may also be used as a solubilizer and wetting agent. Therefore, Labrasol presented in aqueous medium may lower the surface tension, increase the wetting capacity, and make the released drug ease of dissolving or dispersion in micelles. Therefore, the presence of Labrasol as solubilizer and wetting agent may also facilitate the drug solubilization. From this point of view, it is reasonable to find that when the equal amount of Labrasol was replaced by a solid lipid of Precirol ATO 5, not solubilizer and wetting agent, the solubilized drug in the aqueous system decreased in NLC compared with that in ME. The drug solubilized in aqueous phase is of necessary for drug absorption (Mu et al., 2013), especially to the drug being absorbed not via lymphatic route or the dose-dependency in first-pass drug metabolism, the dissolution process shown by in vitro lipid digestion had favorable correlation with in vivo absorption (Dahan and Hoffman, 2006). The higher digestion rate and much more drug partition in the aqueous phase in LT-ME suggested that it may have a faster and higher absorption in vivo than LT-NLC. During the whole reaction, drug content in the pellet phase of LT-NLC was much higher than that in LT-ME (Fig. 8). Additional experiments are needed to determine how the drug content in the pellet phase contributes to drug absorption (Anne et al., 2011). The results of the intestinal absorption of luteolin after lipolysis are shown in Table 4. There was no statistical difference between the Peff values for the two formulations. While the Peff values were significantly larger than those before in vitro lypolysis, indicating that the permeability of both formulations was enhanced after in vitro lypolysis. This may be due to much more drugs were dispersed into the aqueous phase caused by the lypolysis process.
3.6. Bioavailability study To assess the ability of the nanoparticulate carrier system to improve oral bioavailability, the pharmacokinetic parameters of LT-NLC, LT-ME, and LT-suspensions were compared after they were administered to rats by oral gavage. An HPLC method for determination of luteolin concentration in plasma was developed and validated. The intra- and inter-day assay precision were 5.52%, 4.31%, respectively. The absolute and method recovery were 91.78%, 104.97%, respectively. The linear regression equation was A = 41.994C + 0.2346 (r = 0.9995) with the range of 21.2–1590 ng/mL. As illustrated in Fig. 9, after oral administration, the individual mean plasma concentration-time curve profiles of the three different luteolin preparations had a biphasic absorption feature. The pharmacokinetic parameters were calculated with DAS 2.1.1 software using a non-compartmental model. The pharmacokinetic data were shown in Table 5. Tables 6 and 7 indicate that the MRT0–48 h, CLz/F, and AUC0–48 h were statistically different (p < 0.05) between the three luteolin formulations, whereas the Cmax did not change (p = 0.052). The AUC0–48 h of LT-NLC was significantly higher than that of LT-suspension (p < 0.05), whereas there was no difference in the AUC0–48 h between LT-NLC and LT-ME (p = 0.108). The Cmax of LT-ME was significantly higher than those of LT-NLC and LT-suspension, but there was no difference in Cmax between LT-NLC and LT-suspension. The MRT0–48 h of LT-NLC was significantly higher than those of LT-ME and LT-suspension. Next, the relative bioavailability (% Fr) of NLC formulations was calculated using the following equations: %Fr NLC = AUC0 ! 1NLC/AUC0 ! 1suspensions 100% = 515.06%
%Fr ME = AUC0 ! 1ME/AUC0 ! 1suspensions 100% = 885.46% An increase in AUC0–48 h and %Fr for LT-NLC and LT-ME indicated that luteolin oral absorption was significantly enhanced compared to that of LT-suspension. The lower Cmax of LT-NLC indicated that luteolin blood concentration was not too high, as was the case for LT-ME. The in vitro lipolysis data correlated well with the in vivo data. The slower digestion of LT-NLC, described in the in vitro lipolysis results, could lead to decreased absorption in the intestine. Thus, its oral bioavailability was lower than that of LT-ME. It should be mentioned that similar with other lipid carriers, in vivo behavior of NLC and ME are very complex. The factors influencing the Table 6 ANOVA results for the main pharmacokinetic parameters of LT-NLC, LT-ME, and LTsuspension (n = 5).
LT-NLC LT-ME LT-suspension F P
Cmax (ng/mL)
MRT0–48 h (h)
AUC0–48 h (mg h/L)
CLz/F (L/h/kg)
537.152 1594.219 628.902 3.829 0.052
11.614 4.539 2.367 80.555 <0.001
3516.552 6045.463 682.75 6.808 0.011
0.44 0.373 4.404 9.449 0.003
176
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
Table 7 Significance of comparisons between groups (n = 5). Groups
Significance of dependent variable (P)
F1 F1 F2
F2 F3 F3
Cmax (ng/mL)
MRT0–48 h (h)
AUC0–48 h (mg h/L)
CLz/F (L/h/kg)
0.028 0.832 0.042
<0.001 <0.001 0.015
0.108 0.075 0.003
0.951 0.003 0.003
F1, LT-NLC; F2, LT-ME; F3, LT-suspension.
bioavailability of NLC and ME involve many aspects: dispersion process, lipid digestion process, dose-dependency in first-pass drug metabolism, drug efflux by various transports, and so on (Williams et al., 2013; Fricker et al., 2010; Beloqui et al., 2014). In this study, the influence of dispersion process and lipid digestion process to bioavailability were studied for the two preparations, the other factors will be further investigated in future. 4. Conclusions Both NLC and ME improved the oral bioavailability of luteolin, and ME had a higher capacity. LT-ME and LT-NLC had similar properties in the in vitro release and in situ intestinal absorption studies. However, in vitro lipolysis showed that LT-ME is digested faster during the initial stage and the concentration of solubilized drug in the digestion media is higher than that associated with LT-NLC, which may contribute to faster absorption and higher bioavailability. Disclosure There are no conflicts of interest to declare. Acknowledgements This work was financially supported by grant 11ZR1436500 from the Science and Technology Commission of Shanghai Municipality, the National Natural Science Foundation of China (No. 81202925 and No. 81303232), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20123107110005). References Anby, M.U., Williams, H.D., McIntosh, M., Benameur, H., Edwards, G.A., Pouton, C.W., Porter, C.J.H., 2012. Lipid digestion as a trigger for supersaturation: in vitro and in vivo evaluation of the utility of polymeric precipitation inhibitors in selfemulsifying drug delivery systems. Mol. Pharm. 9, 2063–2079. Anne, T.L., Philip, S., Anette, M., 2011. In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems. Int. J. Pharm. 417, 245–255. Bakala N’Goma, J.C., Amara, S., Dridi, K., Jannin, V., Carriere, F., 2012. Understanding lipid digestion in the GI tract for effective drug delivery. Ther. Deliv. 3, 105–124. Beloqui, A., Solinís Á, M., Delgado, A., Evora, C., Isla, A., Rodríguez-Gascón, A., 2014. Fate of nanostructured lipid carriers (NLCs) following the oral route: design, pharmacokinetics and biodistribution. J. Microencaps. 31, 1–8. Bor, J.Y., Chen, H.Y., Yen, G.C., 2006. Evaluation of antioxidant activity and inhibitory effect on nitric oxide production of some common vegetables. J. Agric. Food Chem. 54, 1680–1686. Cershanik, T., Benita, S., 2000. Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs. Eur. J. Pharm. Biopharm. 50, 179–188. Chen, H.Q., Jin, Z.U., Wang, X.J., Xu, X.M., Deng, L., Zhao, J.W., 2008. Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial activation. Neurosci. Lett. 448, 175–179. Constantinides, P.P., 1995. Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharm. Res. 12, 1561–1572. Dahan, A., Hoffman, A., 2006. Use of a dynamic in vitro lipolysis model to rationalize oral formulation development for poor water soluble drugs: correlation with in vivo data and the relationship to intra-enterocyte processes in rats. Pharm. Res. 23, 2165–2174.
Dahan, A., Hoffman, A., Touitou, E., Barry, B.W., 2006. Enhancement in Drug Delivery. CRC Press, pp. 111–127. Devraj, R., Williams, H.D., Warren, D.B., Mohsin, K., Porter, C.J.H., Pouton, C.W., 2013. In vitro assessment of drug-free and fenofibrate-containing lipid formulations using dispersion and digestion testing gives detailed insights into the likely fate of formulations in the intestine. Eur. J. Pharm. Sci. 49, 748–760. Doktorovová, S., Araújo, J., Garcia, M.L., Rakovský, E., Souto, E.B., 2010. Formulating fluticasone propionate in novel PEG-containing nanostructured lipid carriers (PEG-NLC). Colloids Surf. B Biointerfaces 75, 538–542. Fricker, G., Kromp, T., Wendel, A., Blume, A., Zirkel, J., Rebmann, H., Setzer, C., Quinkert, R.O., Setzer, C., Martin, F., Christel, M.G., Müller-Goymann, C., 2010. Phospholipids and lipid-based formulations in oral drug delivery. Pharm. Res. 27, 1469–1486. Günter, S., Irmgard, M., Christoph, M.S., 2008. Anti-oxidant, anti-inflammatory and anti-allergic activities of luteolin. Planta Med. 74, 1667–1677. Hougee, S., Sanders, A., Faber, J., Graus, Y.M., Van den Berg, W.B., Garssen, J., Smit, H. F., Hoijer, M.A., 2005. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages. Biochem. Pharmacol. 69, 241–248. Humberstone, A.J., Charman, W.N., 1997. Lipid-based vehicles for the oral delivery of poorly water soluble drugs. Adv. Drug. Deliv. Rev 25, 103–128. Jores, K., Haberland, A., Wartewig, S., Mäder, K., Mehnert, W., 2005. Solid lipid nanoparticles (SLN) and oil-loaded SLN studied by spectrofluorometry and Raman spectroscopy. Pharm. Res. 22, 1887–1897. Kayoko, S., Noriko, S., Ryushi, N., Motoyoshi, S., Izumi, A., Tsutomu, N., Naohide, K., 2001. Deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation. Drug Metab. Dispos. 29, 1521–1524. Koen, B., Ruth, V., Guido, V., Johannes, V.S., 2005. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. J. Biol. Chem. 280, 5636–5645. Leunga, H.W., Kuob, C.L., Yangc, W.H., Lind, C.H., Lee, H.Z., 2006. Antioxidant enzymes activity involvement in luteolin-induced human lung squamous carcinoma CH27 cell apoptosis. Eur. J. Pharmacol. 534, 12–18. Liu, Y., Wang, P.F., Sun, C., Zhao, J.H., Du, Y., Shi, F., Feng, N.P., 2011. Bioadhesion and enhanced bioavailability by wheat germ agglutinin-grafted lipid nanoparticles for oral delivery of poorly water-soluble drug bufalin. Int. J. Pharm. 31, 260–265. Lu, Q.Z., Yu, A.H., Xi, Y.W., 2009. Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery. Int. J. Pharm. 272, 191–198. Lu, Y., Qi, J.P., Wu, W., 2012. Absorption, distribution and pharmacokinetics of nanoemulsions. Cur. Drug Metab. 13, 396–417. Lu, Y., Wu, K., Li, L., He, Y., Cui, L., Liang, N., Mu, B., 2013. Characterization and evaluation of an oral microemulsion containing the antitumor diterpenoid compound ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid. Int. J. Nanomedicine 8, 1879–1886. Lv, P.C., Li, H.Q., Xue, J.Y., Shi, L., Zhu, H.L., 2009. Synthesis and biological evaluation of novel luteolin derivatives as antibacterial agents. Eur. J. Med. Chem. 44, 908–914. Mu, H.L., Rene, H., Anette, M., 2013. Design of lipid-based formulations for oral administration of poorly water-soluble drug fenofibrate effects of digestion. AAPS Pharm. Sci. Tech. 13, 637–646. Muller, R.H., Radtke, M., Wissing, S.A., 2002. Nanostructured lipid matrices for improved microencapsulation of drugs. Int. J. Pharm. 242, 121–128. Olbrich, C., Kayser, O., Muller, R.H., 2002. Lipase degradation of Dynasan 114 and Dynasan 116 solid lipid nanoparticles (SLN) – effect of surfactants storage time and crystallinity. Int. J. Pharm. 237, 119–128. Paula, L., Hanna, K., Anni, L., 2011. Use of conventional surfactant media as surrogates for FaSSIF in simulating in vivo dissolution of BCS class II drugs. Eur. J. Pharm. Biopharm. 78, 531–538. Porter, C.J.H., Trevaskis, N.L., Charman, W.N., 2007. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat. Rev. Drug Discov. 6, 231–248. Pouton, C.W., 2000. Lipid formulations for oral administration of drugs: nonemulsifying, self-emulsifying and self-microemulsifying drug delivery systems. Eur. J. Pharm. Sci. 11, 93–98. Qi, J.P., Lu, Y., Wu, W., 2012. Absorption, distribution and pharmacokinetics of solid lipid nanoparticles. Cur. Drug Metab. 13, 418–428. Radtke, M., Souro, E.B., Muller, R.H., 2005. Nanostructured lipid carriers:a novel generation of solid lipid drug carriers. Pharm. Technol. Eur. 17, 45–50. Sane, R., Mittapalli, R.K., Elmquist, W.F., 2013. Development and evaluation of a novel microemulsion formulation of elacridar to improve its bioavailability. J. Pharm. Sci. 102, 1343–1354.
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Sek, L., Porter, C.J., Charman, W.N., 2001. Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products by HPTLC coupled with in situ densitometric analysis. Biomed. Anal. 25, 651–661. Shangguan, M., Lu, Y., Qi, J., Han, J., Tian, Z., Xie, Y., Hu, F., Yuan, H., Wu, W., 2014. Binary lipids-based nanostructured lipid carriers for improved oral bioavailability of silymarin. J. Biomater. Appl. 28, 887–896. Thomas, N., Holm, R., Mullertz, A., Rades, T., 2012. In vitro and in vivo performance of novel supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS). J. Control. Release 160, 25–32.
177
Tian, Z.Q., Qi, J.P., i, Y., Han, J., Yuan H. l. Zhang, X., Xie, Y.C., Hu, F.Q., Lu, Y., Wu, W., 2013. Solidification of nanostructured lipid carrier (NLC) onto pellets by fluidbed coating: preparation, in vitro characterization and bioavailability in dogs. Powder Technol. 247, 120–127. Williams, H.D., Trevaskis, N.L., Yeap, Y.Y., Anby, M.U., Pouton, C.W., Porter, C.J., 2013. Lipid-based formulations and drug supersaturation: harnessing the unique benefits of the lipid digestion/absorption pathway. Pharm. Res. 30, 2976–2992. Wu, W., Wang, Y., Que, L., 2006. Enhanced bioavailability of silymarin by selfmicroemulsifying drug delivery system. Eur. J. Pharm. Biopharm. 63, 288–294.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Nanostructured lipid carriers versus microemulsions for delivery of the poorly water-soluble drug luteolin Ying Liu 1, Lan Wang 1, Yiqing Zhao, Man He, Xin Zhang, Mengmeng Niu, Nianping Feng * School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2014 Received in revised form 12 September 2014 Accepted 28 September 2014 Available online 1 October 2014
Objective: Nanostructured lipid carriers and microemulsions effectively deliver poorly water-soluble drugs. However, few studies have investigated their ability and difference in improving drug bioavailability, especially the factors contributed to the difference. Thus, this study was aimed at investigating their efficiency in bioavailability enhancement based on studying two key processes that occur in NLC and ME during traverse along the intestinal tract: the solubilization process and the intestinal permeability process. Methods: The nanostructured lipid carriers and microemulsions had the same composition except that the former were prepared with solid lipids and the latter with liquid lipids; both were evaluated for particle size and zeta potential. Transmission electron microscopy, differential scanning calorimetry, and X-ray diffraction were performed to characterize their properties. Furthermore, in vitro drug release, in situ intestinal absorption, and in vitro lipolysis were studied. The bioavailability of luteolin delivered using nanostructured lipid carriers in rats was compared with that delivered using microemulsions and suspensions. Results: The in vitro analysis revealed different release mechanisms for luteolin in nanostructured lipid carriers and microemulsions, although the in situ intestinal absorption was similar. The in vitro lipolysis data indicated that digestion speed and extent were higher for microemulsions than for nanostructured lipid carriers, and that more of the former partitioned to the aqueous phase. The in vivo bioavailability analysis in rats indicated that the oral absorption and bioavailability of luteolin delivered using nanostructured lipid carriers and microemulsions were higher than those of luteolin suspensions. Conclusion: Nanostructured lipid carriers and microemulsions improved luteolin’s oral bioavailability in rats. The rapid lipid digestion and much more drug solubilized available for absorption in microemulsions may contribute to better absorption and higher bioavailability. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Nanostructured lipids carriers Microemulsions Luteolin In situ intestinal absorption In vitro lipolysis Oral bioavailability
1. Introduction Luteolin (Fig. 1) is a natural 4-dihydroxy flavone compound isolated from Reseda odorata. It has antioxidative, anti-inflammatory/allergic (Bor et al., 2006; Chen et al., 2008; Hougee et al., 2005; Günter et al., 2008; Kayoko et al., 2001), antibacterial (Lv et al., 2009), and anticancer (Koen et al., 2005; Leunga et al., 2006) properties, and has been shown to inhibit protein kinase C
* Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, China. Tel.: +86 21 5132 2198; fax: +86 21 5132 2198. E-mail addresses: [email protected], [email protected] (N. Feng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2014.09.052 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
(Leunga et al., 1989). In clinical application, luteolin is mainly used to treat chronic bronchitis and other respiratory diseases. However, it has very low bioavailability after oral administration because of its poor solubility in aqueous media (Sanghoo et al., 2003). Therefore, there is a clear need to improve the bioavailability of luteolin to increase its potential in clinical application. The most popular approach for improving the oral bioavailability for poorly water-soluble drugs is the use of lipid-based formulations (Pouton, 2000; Humberstone and Charman, 1997; Constantinides, 1995). Lipid vehicles have been used to enhance the absorption of lipophilic drugs for many years. Their mechanisms of action include enhanced dissolution and solubilization of the co-administered lipophilic drug by stimulation of biliary and pancreatic secretions, prolongation of gastrointestinal tract
170
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
OH
OH
O OH
O
OH
Fig. 1. Chemical structure of luteolin.
residence time, stimulation of lymphatic transport, increased intestinal wall permeability, and reduced metabolism and efflux activity (Dahan et al., 2006; Cershanik and Benita, 2000). In the early 1990s, solid lipid nanoparticles (SLN) were developed as an alternative colloidal lipid system for controlled drug delivery. Nanostructured lipid carriers (NLC) composed of a solid lipid matrix with some amount of liquid lipids are new-generation SLN (Muller et al., 2002), which can overcome the limitations of SLN. The presence of liquid lipid creates more imperfections in the matrix to allow accommodation of more drug molecules compared to those in SLN. Furthermore, drug loading is enhanced, drug expulsion during storage is minimized, and the drug release profile can be modulated by varying the lipid matrix (Radtke et al., 2005). Microemulsions, another popular lipid-based delivery approach, are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules. An increasing number of reports suggest that lipid-based microemulsions can be used to enhance the oral bioavailability of drugs, including peptides (Lu et al., 2013; Sane et al., 2013; Wu et al., 2006; Lu et al., 2012). Drug delivery advantages of microemulsions include improved drug solubilization and protection against enzymatic hydrolysis, as well as the potential for enhanced absorption due to surfactant-induced membrane fluidity and permeability changes. There are some research reports on the ability of NLC and microemulsions to enhance the oral bioavailability of poorly water-soluble drugs (Shangguan et al., 2014; Tian et al., 2013; Qi et al., 2012). However, the extent between the two formulations in bioavailability enhancement has not been fully established yet, especially what factors contribute to the difference. After NLC or ME is located within the GI tract, the fate of the loaded drugs is of first essential for efficient absorption, which are determined by the dispersion process and subsequent digestion process (Devraj et al., 2013; Porter et al., 2007). The incorporated drugs are likely to be solubilized within various micelle or micelle similar vehicles, which are formed by the product of lipid digestion to the lipid carriers together with endogenous ingredients such as bile salts and phospholipids. On the other hand, the co-administered drugs have the tendency of precipitation upon loosing the protection of hydrophilic formulation components due to the change of aqueous surroundings during dispersion or lipid digestion process
(Bakala N’Goma et al., 2012; Anby et al., 2012; Thomas et al., 2012). Due to lipid components and the surfactants containing ester groups from NLC and ME are all the substrates of the hydrolysis reaction by pancreatic lipase, it is rational to deduce that lipid digestion process is inevitable in vivo, which may influence drug absorption and bioavailability. Thus, the aim of present study was to evaluate their efficiency in bioavailability enhancement based on the investigation of two key processes that occur in NLC and ME during traverse along the intestinal tract: the solubilization process and the intestinal permeability process. A dynamic in vitro lipolysis assay was used to determine drug solubilization, and intestinal permeability was tested with an in situ rat intestinal absorption model. The results of in vitro and in situ analyses were subsequently compared with in vivo oral bioavailability data. 2. Materials and methods 2.1. Materials Luteolin was obtained from Jiangxi Bencaotiangong Medical Technology Co., Ltd., China. Glyceryl palmitostearate (Precirol ATO1 5) and caprylocaproyl macrogol-8 glycerides (Labrasol1) were donated by Gattefossè, France. Polyoxyl castor oil (Cremophor ELP) was donated by BASF, Germany. Tris maleate, sodium taurodeoxycholate (NaTDC), and porcine pancreatin were purchased from Sigma–Aldrich, USA. 4-Bromophenylboronic acid (4-BPB) was purchased from Acros Organics, Belgium. Phosphatidylcholine (PC) was purchased from Lipoid, Germany. Methanol (HPLC grade) was purchased from Merck, Germany. Phosphoric acid, perchlorate, ethyl acetate, glucose, mannose, sucrose, lactose, and mycose were purchased from Shanghai Chemical Reagent Co., China. All other chemicals were HPLC or analytical grade. 2.2. Preparation of luteolin-NLC (LT-NLC) and luteolin-ME (LT-ME) The composition of the two preparations is shown in Table 1. Hot high-pressure homogenization (HPH) (GEA Niro Soavi NS1001L, Homogenizer systems, Italy) was used to prepare LT-NLC. Briefly, solid lipid, liquid lipid, and surfactant were heated at 70 C with continuous stirring to homogeneously mix the lipids. The drug was dissolved in the melted mixture. Then, distilled water heated to 70 C was poured into the mixture and homogeneously dispersed using a homogenizer (IKA1 RCT, Germany) at 10,000 rpm for 10 min, while maintaining the temperature at 70 C. This pre-emulsion was further homogenized for five cycles at 800 bar. The dispersion was immediately placed in an ice bath with gentle stirring for 10 min before further use. LT-ME was prepared by dissolving luteolin in a mixture of Labrasol and Cremophor ELP. Then, distilled water was dropped into the mixture with gentle stirring until uniform and transparent preparations were formed. 2.3. Determination of particle size, polydispersity index (PDI), and zeta potential Particle size (z-average diameter) and PDI were measured with a dynamic light scattering technique using a Malvern Zetasizer
Table 1 Formulation composition of LT-NLC and LT-ME. Formulation
Ingredient (%, w/w) Luteolin
Precirol ATO 5
Labrasol
Cremophor ELP
Distilled water
LT-NLC LT-ME
0.01 0.01
3.48 –
0.75 4.23
2.61 2.61
93.15 93.15
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Table 2 Particle size, PDI, and zeta potential of LT-NLC and LT-ME.
LT-NLC LT-ME
Particle size/nm
PDI
Zeta potential/mv
43.89 4.52 12.98 0.14
0.219 0.007 0.202 0.038
19.42 0.79 4.39 0.32
(n = 3).
Nano ZS90 (Malvern Instruments, UK) at 25 C and an angle of 90 . Zeta potential was measured with the same instrument. Before measurement, the preparations were diluted with deionized water to achieve a suitable scattering intensity. Each sample was analyzed in triplicate. 2.4. Characterization of LT-NLC 2.4.1. Determination of drug loading and encapsulation efficiency Drug loading and encapsulation efficiency were determined using a previously reported method (Lu et al., 2009). Briefly, after
171
filtration through a 0.45-mm filter (Sartorius, Germany), the filtrate was diluted 100-fold with acetone. It was mixed well using a cyclomixer to completely extract the drug from lipid to acetone. Then, the drug content was analyzed by HPLC. The amount of soluble unencapsulated drug was measured by ultrafiltration using centrifugal filter tubes with a molecular weight cut-off of 300 kDa (Millipore, Ireland). Briefly, a 200-mL aliquot of LT-NLC was placed into the centrifugal filter tube and centrifuged at 5827 g for 10 min. The amount of drug in the aqueous phase was determined by HPLC. Drug loading and encapsulation efficiency were calculated using Eqs. (1) and (2), respectively. Load contentð%Þ ¼
Ws 100% W lipid
Entrapment efficiencyð%Þ ¼
Ws 100% W total
(1)
(2)
where Ws represents the amount of luteolin in the NLC, Wlipid is the weight of the vehicle, and Wtotal represents the amount of luteolin used in the formulation. The HPLC assay was performed as follows. A reverse phase C18 column (Diamond C18, 250 mm 4.6 mm, 5 mm; Dikma) was used. A mixture of methanol (40%) and 0.1% aqueous solution of phosphoric acid (60%) was used as the mobile phase. Column temperature, mobile phase flow rate, injection volume, and detection wavelength were set at 30 C, 1 mL/min, 20 mL, and 348 nm, respectively. The assay method was validated and the calibration curve was linear (r = 0.9999) within the 0.60–39.96 mg/ mL range. 2.4.2. Transmission electron microscopy (TEM) The morphology of NLC was determined by TEM (JEM-1230, JEOL, Japan). The samples were diluted with distilled water, and then stained with 1% (w/v) phosphotungstic acid and placed on copper grids with films. After air-drying for 3 h at room temperature, the specimens were analyzed by TEM. 2.4.3. Differential scanning calorimetry (DSC) analysis NLC dispersions were lyophilized (Alpha 1-4, Marein Christ, Germany) prior to DSC analysis. Solid lipid (Precirol ATO 5), drug (luteolin), physical lipid/drug mixture, cryoprotectant mixture (mannose, mycose), and drug-loaded NLC were subjected to DSC (DSC 204 F1 Phoenix1, Netzsch, Germany) analysis. Briefly, samples were kept in standard aluminum pans and sealed. DSC analysis was performed at 10 C/min at temperatures of 30 to 350 C in a nitrogen environment. An empty, sealed pan was used as a reference. The thermograms were recorded.
Fig. 2. Transmission electron micrograph of (A) blank-NLC and (B) LT-NLC.
Fig. 3. DSC curve of (A)LT-NLC; (B) physical mixture; (C) mixture of cyroprotectant; (D) Precirol ATO 5; (E) luteolin.
172
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
Q ½1 Ph:redout =Ph:redin L
2.4.4. X-ray diffraction (XRD) analysis XRD analysis was conducted using a powder X-ray diffractometer (D/MAX 2550 VB/PC, Rigaku, Japan), and Cu/Ka radiation was used as the X-ray source. Samples were placed in the glass holders and scanned from 3 to 50 with a scan angular speed (2u/min) of 8 /min at an operating voltage of 40 kV and a current of 100 mA.
NWF ¼
2.5. In vitro release
Ph. redout and Ph. redin are the outlet and inlet concentrations of the non-absorbable water flux marker phenol red, Cin is the inlet concentration, and Cout is the outlet concentration of compound, which is corrected for volume change in the segment by using the phenol red concentration in the outlet and inlet tubing. Q is the flow rate (0.2 mL/min), L is the length of the segment, and r is the rat intestinal radius.
In vitro release studies were carried out (Liu et al., 2011) in 100 mL of a release medium composed of PBS (pH 7.4) containing 0.5% Tween 80 at 37 C. Preparations (4 mL) were added to a pretreated dialysis bag, then they were immersed in a shaker water bath with shaking speed of 100 strokes/min. One milliliter of the release medium was removed at predetermined time intervals, and the volume was replaced by adding 1 mL of fresh medium. Luteolin concentration in the removed release medium was measured by HPLC. 2.6. In situ intestinal absorption study The experiment was approved by the institutional animal care and use committee, Shanghai University of T.C.M. Adult male Sprague-Dawley rats (weight, 300 20 g) were obtained from the shanghai laboratory animal center. They were housed in air-conditioned quarters with a 12-h light/12-h dark photoperiod schedule. The animals received a standard laboratory diet and tap water ad libitum. The rats were randomly divided into two groups (NLC and ME). In each group, three concentrations of luteolin (2, 5, and 10 mg/mL) were used in separate experiments. The anesthetized rats were restrained in a supine position on a board with temperature maintained at 37 C. A small midline incision was made in the abdomen and a 10-cm section of the small intestine was cannulated. Blank perfusion buffer was infused for 10 min by using a syringe pump followed by perfusion with different concentrations (2, 5, and 10 mg/mL) of luteolin at a flow rate of 0.2 mL/min for 90 min. Outlet samples were collected in microtubes at appropriate intervals (30, 40, 50, 60, 70, and 80 min). The sample volume for each time interval was 2 mL. The length and the radius of segment were measured when the experiment was completed. Samples were stored at 20 C until analysis. Samples from the perfusion study were filtered and directly injected into the HPLC column and required no further preparation prior to analysis. Effective permeability coefficients (Peff) were calculated from the steady-state concentrations of compounds in the collected perfusate, which were considered to be reached when the concentration of phenol red was at a steady state. Concentrations reached a steady state about 40 min after the start of perfusion, which was confirmed by plotting the ratio of the outlet to inlet concentration (corrected for water transport) versus time. The intestinal net water flux (NWF, mL/h/cm) and Peff were calculated using Eqs. (3) and (4), respectively.
Fig. 4. X-ray-diffraction patterns of (A) physical mixture; (B) cryoprotectant; (C) Precirol ATO 5; (D) LT-NLC; (E) blank-NLC; (F) luteolin.
Peff ¼
Q lnðC in =C out Þ 2p rL
(3)
(4)
2.7. In vitro lipolysis study The dynamic in vitro lipolysis experiment was designed to achieve pseudo-physiological conditions. The in vitro lipolysis study was carried out as previously reported (Dahan and Hoffman, 2006). The experimental medium, comprising 29.25 mL of digestion buffer (50 mM Tris maleate, 150 mM NaCl, and 5 mM CaCl2, pH 7.5) containing 5 mM taurocholic acid and 1.25 mM PC (conditions mimicking fasted state GIT), was continuously stirred at 100 rpm and maintained at 37 C. Then, 6.75 mL of the test formulation (NLC or ME, stirred and gently heated to 37 C, and freshly prepared 30 min before each experiment) was dispersed in the medium and stirred for 15 min. Fresh pancreatin extract was prepared by adding 1 g of porcine pancreatin powder to 5 mL of digestion buffer, stirring for 15 min, and centrifuging as previously described (Sek et al., 2001). Approximately 4 mL of the pancreatin extract (containing 40,000 tributyrin units of pancreatic lipase activity) was added to the medium to initiate the enzymatic digestion of the formulation. A pH titrator unit (TitraLab 856 pH-Stat, Radiometer, France) was used to maintain the pH at 7.5 throughout the experiment. The experiment was continued for 60 min, in which time the enzymatic digestion process was completed, as indicated by the completion of the pH titration. The intestinal absorption of luteolin after lipolysis was also determined. Medium from the in vitro lipolysis experiment, collected immediately after the completion of the lipolysis process, was added to the perfusion buffer to start the in situ intestinal absorption experiment. 2.8. In vivo bioavailability The animal experiments were approved by the institutional animal ethical committee of Shanghai University of TCM. Male Sprague-Dawley rats (weight, 300 20 g) were randomly divided into 3 groups of 5 animals each. Prior to drug administration, the
Fig. 5. In vitro release curves of luteolin from LT-NLC and LT-ME.
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Table 3 Permeability coefficients (Peff) for LT-NLC and LT-ME determined in rats (n = 3). Group
Cin (mg/mL)
Mean Peff (103 cm/min)
Mean NWF (mL/min/cm)
LT-NLC
2 5 10 2 5 10
2.57 0.97 4.78 2.74 5.10 3.04 2.38 1.61 6.36 2.50 12.70 4.79
0.00304 0.000787 0.00039 0.001146 0.00062 0.001951 0.00057 0.000458 0.00259 0.001019 0.00014 0.003642
LT-ME
animals were fasted for 12 h with free access to water. The test preparations were LT-NLC, LT-ME, and LT-suspensions. LT-ME and LT-NLC were prepared as described in Section 2.2. LT-suspensions were prepared by dispersing luteolin in sodium carboxymethyl cellulose (CMC-Na) solution containing glycerin. The rats were administered 3.46 mg/kg luteolin with each preparation by oral gavage. Blood samples (1 mL) were collected into heparinized tubes by retro-orbital puncture 0, 0.017, 0.25, 0.5, 1, 2, 3, 5, 8, 12, 24, and 48 h after treatment. Blood samples were centrifuged at 8000 rpm for 5 min. Plasma was kept at 20 C until analysis as described below. Two-hundred microliters of 6.0% perchloric acid was added slowly to 200 mL of plasma to precipitate protein completely. Luteolin was extracted with 3.0 mL of ethyl acetate by vortexing for 5 min. After centrifugation at 8000 rpm for 10 min, 2.40 mL of the organic phase supernatant was carefully transferred to another tube and nitrogen flow-dried. The residue was reconstituted in 100 mL of the mobile phase; the mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was injected into the HPLC system. HPLC analysis was performed on a Diamond C18 column (250 mm 4.6 mm, 5 mm). Optimum separation was carried out with a mobile phase composed of methanol and 0.1% phosphoric acid aqueous solution (55:45, v/v) at a flow rate of 1.0 mL/min. The injected volume of sample was 30 mL, and the detection wavelength and column temperature were set at 348 nm and 30 C, respectively.
173
3. Results and discussion 3.1. Particle size, PDI, and zeta potential The mean particle size of LT-NLC and LT-ME is shown in Table 2. Both preparations had a particle size of less than 50 nm with a low PDI. The ME had a smaller size of 12.98 nm than the NLC (43.89 nm), which may be due to the formation composition difference in oil phase. In the case of NLC, Precirol ATO 5 (glyceryl palmitostearate) was used as solid lipid of NLC, and formed its core. The liquid lipid of labrasol was reported to be well entrapped within solid lipid matrix of Precirol ATO-5 (Doktorovová et al., 2010). In addition, the liquid lipid would be located near or at the surface of lipid nanoparticles, as suggested by Jores et al. (2005) which attributes to physical characteristics such as particle size and zeta potential. While the lipid of ME was totally composed by Labrasol (PEG-6 caprybic/capric triglycerides). Labrasol, except application as lipid of nanocarrier, it is often used as solubilizer and wetting agents, which has relative stronger ability to be dispersed in aqueous surroundings, and therefore attributed to smaller particle size of ME. 3.2. Characterization of LT-NLC
Pharmacokinetic parameters were determined using DAS 2.1.1 software. Statistical analysis for the pharmacokinetic parameters was carried out using one-way analysis of variance (ANOVA). A p value of <0.05 was considered statistically significant.
The drug loading and encapsulation efficiency of the LT-NLC were 0.15% and 95.63%, respectively. The morphology of NLC determined by TEM is shown in Fig. 2. The particles had almost spherical and uniform shapes and were well dispersed. DSC was used to investigate the drug state and crystallization in the compounds and NLC by determining the variations of temperature and energy at phase transition. Fig. 3 shows the DSC curves of Precirol ATO 5, luteolin, cryoprotectant mixture, their physical mixture, and drug-loaded NLC lyophilized powder. The thermogram of luteolin showed a melting peak at 307.5 C. The melting peak for Precirol ATO 5 was observed at 60.4 C, and those for the cryoprotectant mixture at 101.0 and 168.5 C. The melting peaks of luteolin were distinct, whereas those of Precirol ATO 5 and the cryoprotectant mixture had almost same value in the curve as the physical mixture. During the heating process, luteolin was dissolved in the melted solid lipid, which might have led to the disappearance of the peak. With the exception of Precirol ATO 5, all endothermic peaks disappeared in LT-NLC powder, and a new peak appeared at 152.9 C, which suggested that luteolin in the NLC was in an amorphous rather than a crystalline state, to form a solid solution within the matrix of nanoparticles.
Fig. 6. In vitro lipolysis rate of LT-NLC and LT-ME.
Fig. 7. Partition of luteolin in aqueous phase after in vitro lipolysis.
2.9. Data analysis
174
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
effect of drug diffusion was predominant and a smaller amount of drug was retained in the core of the NLC than in the shell. The release profile of LT-ME was fitted to the Higuchi model and the equation was as follows: F = 1.214 t1/2 (r = 0.9665). The results indicated that the release mechanisms of LT-NLC and LT-ME were different. 3.4. In situ intestinal absorption study
Fig. 8. Partition of luteolin in the pellet phase after in vitro lipolysis.
XRD was used to identify the physical state of luteolin incorporated in NLC. The patterns of solid lipid (Precirol ATO 5), drug (luteolin), physical mixture (lipid, drug, and cryoprotectant mixture), cryoprotectant mixture (mannose, mycose), drug-loaded NLC, and drug-free NLC are shown in Fig. 4. For pure luteolin, significant diffraction peaks, indicating that luteolin was in the crystalline form, were detected at 2u scattered angles of 6.879 , 13.561, 13.840 , 14.540 , and 16.280 . The solid–lipid (Precirol ATO 5) pattern showed two characteristic wide peaks (5.260 and 21.460 ), indicating that the bulk matrix (Precirol ATO 5) was also in the crystalline form. There was no distinct peak for LT-NLC, suggesting that it was not crystalized. Some characteristic peaks of Precirol ATO 5 in NLC disappeared and the position of the 21.460 peak shifted forward and its intensity decreased. The results suggest that luteolin was in the amorphous, rather than crystalline state in NLC, and the bulk matrix (Precirol ATO 5) was in a lesserorder crystal. 3.3. In vitro release We used the dialysis bag diffusion technique to investigate luteolin release from LT-NLC and LT-ME in PBS (pH 7.4). The mean cumulative percent release versus time curves are shown in Fig. 5. Compared to the release of LT-ME, the cumulative percent release of LT-NLC in PBS at 0.5 h was approximately 14%. LT-NLC showed an initial burst release, suggesting that there was unencapsulated drug attached to the surface of the particles. A sustained release profile was observed for both LT-NLC and LT-ME, which could increase the circulation time of luteolin, leading to prolonged drug residence time in the systemic circulation and better bioavailability. The release profile of the drug from NLC in PBS was fitted to the Ritger-Peppa’s kinetics model and the equation was as follows: F = 2.369 t0.433 (r = 0.9859). Based on the model, the value of n was 0.433 (0.43 < n < 1.0), suggesting that luteolin release from NLC was because of drug diffusion and lipid matrix erosion. The n value was close to 0.45, indicating that the
The drug amount and total volume of perfusate changed continuously during the experiment because of the absorption in the GI tract. Therefore, to determine the volume change of the perfusate in the intestinal perfusion experiment, phenol red was added into the perfusate solution as a non-absorbed marker. The mean Peff values for different concentrations of LT-NLC and LT-ME as well as the mean net water fluxes, determined with the single pass intestinal perfusion technique are listed in Table 3. The Peff values for different concentrations of LT-NLC were not statistically different (p = 0.433). However, there were differences between the Peff values for different concentrations of LT-ME (p = 0.022). This may be because of the presence of the surfactant in LT-ME. The compositions of LT-ME and LT-NLC developed in this study were similar, except that the former contained liquid and the latter solid lipid. The liquid lipid used was Labrasol, a caprylocaproyl macrogolglyceride, which had a higher HLB value (14), indicating that it had surfactant properties, which could affect permeability. However, further investigation is necessary to elucidate the exact mechanism. There were no differences between the following concentrations of LT-NLC and LT-ME: 2 mg/mL (p = 0.868), 5 mg/mL (p = 0.503), and 10 mg/mL (p = 0.081). 3.5. In vitro lipolysis study During the process of in vitro lipolysis of lipid-based formulations, free fatty acids are formed as a result of lipid degradation. The release of the fatty acids caused a decrease in pH, and NaOH (0.2 M) was added to keep the pH value constant. As a result, NaOH decrease was used to estimate the degradation velocity of the lipid by pancreatic lipase (Fig. 6). In the first 5 min, there was a high initial decrease of NaOH, corresponding to a high output of free fatty acids. The titration curves indicated that the digestion speed was higher for LT-ME than for LT-NLC in the first 20 min, and subsequently LT-ME degradation was slower than that of LT-NLC. This result may be because of the solid lipid present in NLC. Previous studies have shown that solid lipids are still digested by lipase, but the digestion rate is lower than that of liquid lipids (Olbrich et al., 2002). In this study, the existence of solid lipid in NLC may lead to a slower digestion rate compared to that of ME.
Table 4 T-test for LT-NLC and LT-ME Peff values after digestion.
LT-NLC LT-ME F P
Mean Peff (103 cm/min)
Mean NWF (mL/min/cm)
54.36 4.97 64.55 6.55 4.615 0.098
0.00247 0.000979 0.002723 0.00198 – –
Fig. 9. Plasma concentration profile of luteolin after oral administration of LT-NLC, LT-ME and LT-suspensions in rats (n = 5).
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
175
Table 5 Pharmacokinetic parameters of luteolin after oral administration of LT-NLC, LT-ME, and LT-suspensions in rats (n = 5).
LT-NLC LT-ME LT-suspension
AUC0–t (mg h/L)
AUC0–1(mg h/L)
MRT0–t (h)
T1/2z (h)
Tmax (h)
CLz/F (L/h/kg)
3516.552 6045.463 682.75
10663.64 16222.6 1152.713
11.614 4.539 2.367
9.912 4.501 1.238
2.6 4.6 0.25
0.44 0.373 4.404
During lipolysis, samples were collected at specific time points and the lipase activity was inhibited immediately with 4-bromobenzeneboronic acid. Each sample was ultracentrifuged and three phases were formed: (1) the oil phase, (2) the aqueous phase, and (3) the pellet. The aqueous phase is of particular interest for drug solubilization in the GI tract, because drug absorption is believed to happen during this phase (Mu et al., 2013). The results of luteolin partitioning in the aqueous phase after in vitro lipolysis of LT-NLC and LT-ME are shown in Fig. 7. The percentage of Luteolin participated in the aqueous phase increased gradually for both NLC and ME. The product of lipid digestion with bile salts and phospholipids form micelles, or mixed micelles, or other lipid vehicles, in which drug may be incorporated into these colloidal systems, and thus facilitate drug solubilization. In the first 30 min, drug content in the aqueous phase was higher for LT-NLC than for LT-ME, while after 45 min, the pattern reversed. In the case of NLC, part of Luteolin may be absorbed or locate at the surface of NLC, burst release occurred upon NLC dispersion into aqueous media as shown in Fig. 5. This burst release phenomenon may contribute to the higher drug content in the aqueous phase for LT-NLC than for LT-ME in the first 30 min. Labrasol was used as a liquid oil in NLC and ME formulation. Due to the lipid hydrolysis by pancreatic lipase, the lipolytic products together with other components such as bile salts and phospholipids may form micelles, which may increase the solubilized drug in the system. In addition, the properties of dispersion media such as CMC, surface tension, diffusion capacity have been shown to influence drug dissolve in aqueous media (Paula et al., 2011). Labrasol as a pharmaceutical excipient may also be used as a solubilizer and wetting agent. Therefore, Labrasol presented in aqueous medium may lower the surface tension, increase the wetting capacity, and make the released drug ease of dissolving or dispersion in micelles. Therefore, the presence of Labrasol as solubilizer and wetting agent may also facilitate the drug solubilization. From this point of view, it is reasonable to find that when the equal amount of Labrasol was replaced by a solid lipid of Precirol ATO 5, not solubilizer and wetting agent, the solubilized drug in the aqueous system decreased in NLC compared with that in ME. The drug solubilized in aqueous phase is of necessary for drug absorption (Mu et al., 2013), especially to the drug being absorbed not via lymphatic route or the dose-dependency in first-pass drug metabolism, the dissolution process shown by in vitro lipid digestion had favorable correlation with in vivo absorption (Dahan and Hoffman, 2006). The higher digestion rate and much more drug partition in the aqueous phase in LT-ME suggested that it may have a faster and higher absorption in vivo than LT-NLC. During the whole reaction, drug content in the pellet phase of LT-NLC was much higher than that in LT-ME (Fig. 8). Additional experiments are needed to determine how the drug content in the pellet phase contributes to drug absorption (Anne et al., 2011). The results of the intestinal absorption of luteolin after lipolysis are shown in Table 4. There was no statistical difference between the Peff values for the two formulations. While the Peff values were significantly larger than those before in vitro lypolysis, indicating that the permeability of both formulations was enhanced after in vitro lypolysis. This may be due to much more drugs were dispersed into the aqueous phase caused by the lypolysis process.
3.6. Bioavailability study To assess the ability of the nanoparticulate carrier system to improve oral bioavailability, the pharmacokinetic parameters of LT-NLC, LT-ME, and LT-suspensions were compared after they were administered to rats by oral gavage. An HPLC method for determination of luteolin concentration in plasma was developed and validated. The intra- and inter-day assay precision were 5.52%, 4.31%, respectively. The absolute and method recovery were 91.78%, 104.97%, respectively. The linear regression equation was A = 41.994C + 0.2346 (r = 0.9995) with the range of 21.2–1590 ng/mL. As illustrated in Fig. 9, after oral administration, the individual mean plasma concentration-time curve profiles of the three different luteolin preparations had a biphasic absorption feature. The pharmacokinetic parameters were calculated with DAS 2.1.1 software using a non-compartmental model. The pharmacokinetic data were shown in Table 5. Tables 6 and 7 indicate that the MRT0–48 h, CLz/F, and AUC0–48 h were statistically different (p < 0.05) between the three luteolin formulations, whereas the Cmax did not change (p = 0.052). The AUC0–48 h of LT-NLC was significantly higher than that of LT-suspension (p < 0.05), whereas there was no difference in the AUC0–48 h between LT-NLC and LT-ME (p = 0.108). The Cmax of LT-ME was significantly higher than those of LT-NLC and LT-suspension, but there was no difference in Cmax between LT-NLC and LT-suspension. The MRT0–48 h of LT-NLC was significantly higher than those of LT-ME and LT-suspension. Next, the relative bioavailability (% Fr) of NLC formulations was calculated using the following equations: %Fr NLC = AUC0 ! 1NLC/AUC0 ! 1suspensions 100% = 515.06%
%Fr ME = AUC0 ! 1ME/AUC0 ! 1suspensions 100% = 885.46% An increase in AUC0–48 h and %Fr for LT-NLC and LT-ME indicated that luteolin oral absorption was significantly enhanced compared to that of LT-suspension. The lower Cmax of LT-NLC indicated that luteolin blood concentration was not too high, as was the case for LT-ME. The in vitro lipolysis data correlated well with the in vivo data. The slower digestion of LT-NLC, described in the in vitro lipolysis results, could lead to decreased absorption in the intestine. Thus, its oral bioavailability was lower than that of LT-ME. It should be mentioned that similar with other lipid carriers, in vivo behavior of NLC and ME are very complex. The factors influencing the Table 6 ANOVA results for the main pharmacokinetic parameters of LT-NLC, LT-ME, and LTsuspension (n = 5).
LT-NLC LT-ME LT-suspension F P
Cmax (ng/mL)
MRT0–48 h (h)
AUC0–48 h (mg h/L)
CLz/F (L/h/kg)
537.152 1594.219 628.902 3.829 0.052
11.614 4.539 2.367 80.555 <0.001
3516.552 6045.463 682.75 6.808 0.011
0.44 0.373 4.404 9.449 0.003
176
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177
Table 7 Significance of comparisons between groups (n = 5). Groups
Significance of dependent variable (P)
F1 F1 F2
F2 F3 F3
Cmax (ng/mL)
MRT0–48 h (h)
AUC0–48 h (mg h/L)
CLz/F (L/h/kg)
0.028 0.832 0.042
<0.001 <0.001 0.015
0.108 0.075 0.003
0.951 0.003 0.003
F1, LT-NLC; F2, LT-ME; F3, LT-suspension.
bioavailability of NLC and ME involve many aspects: dispersion process, lipid digestion process, dose-dependency in first-pass drug metabolism, drug efflux by various transports, and so on (Williams et al., 2013; Fricker et al., 2010; Beloqui et al., 2014). In this study, the influence of dispersion process and lipid digestion process to bioavailability were studied for the two preparations, the other factors will be further investigated in future. 4. Conclusions Both NLC and ME improved the oral bioavailability of luteolin, and ME had a higher capacity. LT-ME and LT-NLC had similar properties in the in vitro release and in situ intestinal absorption studies. However, in vitro lipolysis showed that LT-ME is digested faster during the initial stage and the concentration of solubilized drug in the digestion media is higher than that associated with LT-NLC, which may contribute to faster absorption and higher bioavailability. Disclosure There are no conflicts of interest to declare. Acknowledgements This work was financially supported by grant 11ZR1436500 from the Science and Technology Commission of Shanghai Municipality, the National Natural Science Foundation of China (No. 81202925 and No. 81303232), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20123107110005). References Anby, M.U., Williams, H.D., McIntosh, M., Benameur, H., Edwards, G.A., Pouton, C.W., Porter, C.J.H., 2012. Lipid digestion as a trigger for supersaturation: in vitro and in vivo evaluation of the utility of polymeric precipitation inhibitors in selfemulsifying drug delivery systems. Mol. Pharm. 9, 2063–2079. Anne, T.L., Philip, S., Anette, M., 2011. In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems. Int. J. Pharm. 417, 245–255. Bakala N’Goma, J.C., Amara, S., Dridi, K., Jannin, V., Carriere, F., 2012. Understanding lipid digestion in the GI tract for effective drug delivery. Ther. Deliv. 3, 105–124. Beloqui, A., Solinís Á, M., Delgado, A., Evora, C., Isla, A., Rodríguez-Gascón, A., 2014. Fate of nanostructured lipid carriers (NLCs) following the oral route: design, pharmacokinetics and biodistribution. J. Microencaps. 31, 1–8. Bor, J.Y., Chen, H.Y., Yen, G.C., 2006. Evaluation of antioxidant activity and inhibitory effect on nitric oxide production of some common vegetables. J. Agric. Food Chem. 54, 1680–1686. Cershanik, T., Benita, S., 2000. Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs. Eur. J. Pharm. Biopharm. 50, 179–188. Chen, H.Q., Jin, Z.U., Wang, X.J., Xu, X.M., Deng, L., Zhao, J.W., 2008. Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial activation. Neurosci. Lett. 448, 175–179. Constantinides, P.P., 1995. Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharm. Res. 12, 1561–1572. Dahan, A., Hoffman, A., 2006. Use of a dynamic in vitro lipolysis model to rationalize oral formulation development for poor water soluble drugs: correlation with in vivo data and the relationship to intra-enterocyte processes in rats. Pharm. Res. 23, 2165–2174.
Dahan, A., Hoffman, A., Touitou, E., Barry, B.W., 2006. Enhancement in Drug Delivery. CRC Press, pp. 111–127. Devraj, R., Williams, H.D., Warren, D.B., Mohsin, K., Porter, C.J.H., Pouton, C.W., 2013. In vitro assessment of drug-free and fenofibrate-containing lipid formulations using dispersion and digestion testing gives detailed insights into the likely fate of formulations in the intestine. Eur. J. Pharm. Sci. 49, 748–760. Doktorovová, S., Araújo, J., Garcia, M.L., Rakovský, E., Souto, E.B., 2010. Formulating fluticasone propionate in novel PEG-containing nanostructured lipid carriers (PEG-NLC). Colloids Surf. B Biointerfaces 75, 538–542. Fricker, G., Kromp, T., Wendel, A., Blume, A., Zirkel, J., Rebmann, H., Setzer, C., Quinkert, R.O., Setzer, C., Martin, F., Christel, M.G., Müller-Goymann, C., 2010. Phospholipids and lipid-based formulations in oral drug delivery. Pharm. Res. 27, 1469–1486. Günter, S., Irmgard, M., Christoph, M.S., 2008. Anti-oxidant, anti-inflammatory and anti-allergic activities of luteolin. Planta Med. 74, 1667–1677. Hougee, S., Sanders, A., Faber, J., Graus, Y.M., Van den Berg, W.B., Garssen, J., Smit, H. F., Hoijer, M.A., 2005. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages. Biochem. Pharmacol. 69, 241–248. Humberstone, A.J., Charman, W.N., 1997. Lipid-based vehicles for the oral delivery of poorly water soluble drugs. Adv. Drug. Deliv. Rev 25, 103–128. Jores, K., Haberland, A., Wartewig, S., Mäder, K., Mehnert, W., 2005. Solid lipid nanoparticles (SLN) and oil-loaded SLN studied by spectrofluorometry and Raman spectroscopy. Pharm. Res. 22, 1887–1897. Kayoko, S., Noriko, S., Ryushi, N., Motoyoshi, S., Izumi, A., Tsutomu, N., Naohide, K., 2001. Deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation. Drug Metab. Dispos. 29, 1521–1524. Koen, B., Ruth, V., Guido, V., Johannes, V.S., 2005. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. J. Biol. Chem. 280, 5636–5645. Leunga, H.W., Kuob, C.L., Yangc, W.H., Lind, C.H., Lee, H.Z., 2006. Antioxidant enzymes activity involvement in luteolin-induced human lung squamous carcinoma CH27 cell apoptosis. Eur. J. Pharmacol. 534, 12–18. Liu, Y., Wang, P.F., Sun, C., Zhao, J.H., Du, Y., Shi, F., Feng, N.P., 2011. Bioadhesion and enhanced bioavailability by wheat germ agglutinin-grafted lipid nanoparticles for oral delivery of poorly water-soluble drug bufalin. Int. J. Pharm. 31, 260–265. Lu, Q.Z., Yu, A.H., Xi, Y.W., 2009. Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery. Int. J. Pharm. 272, 191–198. Lu, Y., Qi, J.P., Wu, W., 2012. Absorption, distribution and pharmacokinetics of nanoemulsions. Cur. Drug Metab. 13, 396–417. Lu, Y., Wu, K., Li, L., He, Y., Cui, L., Liang, N., Mu, B., 2013. Characterization and evaluation of an oral microemulsion containing the antitumor diterpenoid compound ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid. Int. J. Nanomedicine 8, 1879–1886. Lv, P.C., Li, H.Q., Xue, J.Y., Shi, L., Zhu, H.L., 2009. Synthesis and biological evaluation of novel luteolin derivatives as antibacterial agents. Eur. J. Med. Chem. 44, 908–914. Mu, H.L., Rene, H., Anette, M., 2013. Design of lipid-based formulations for oral administration of poorly water-soluble drug fenofibrate effects of digestion. AAPS Pharm. Sci. Tech. 13, 637–646. Muller, R.H., Radtke, M., Wissing, S.A., 2002. Nanostructured lipid matrices for improved microencapsulation of drugs. Int. J. Pharm. 242, 121–128. Olbrich, C., Kayser, O., Muller, R.H., 2002. Lipase degradation of Dynasan 114 and Dynasan 116 solid lipid nanoparticles (SLN) – effect of surfactants storage time and crystallinity. Int. J. Pharm. 237, 119–128. Paula, L., Hanna, K., Anni, L., 2011. Use of conventional surfactant media as surrogates for FaSSIF in simulating in vivo dissolution of BCS class II drugs. Eur. J. Pharm. Biopharm. 78, 531–538. Porter, C.J.H., Trevaskis, N.L., Charman, W.N., 2007. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat. Rev. Drug Discov. 6, 231–248. Pouton, C.W., 2000. Lipid formulations for oral administration of drugs: nonemulsifying, self-emulsifying and self-microemulsifying drug delivery systems. Eur. J. Pharm. Sci. 11, 93–98. Qi, J.P., Lu, Y., Wu, W., 2012. Absorption, distribution and pharmacokinetics of solid lipid nanoparticles. Cur. Drug Metab. 13, 418–428. Radtke, M., Souro, E.B., Muller, R.H., 2005. Nanostructured lipid carriers:a novel generation of solid lipid drug carriers. Pharm. Technol. Eur. 17, 45–50. Sane, R., Mittapalli, R.K., Elmquist, W.F., 2013. Development and evaluation of a novel microemulsion formulation of elacridar to improve its bioavailability. J. Pharm. Sci. 102, 1343–1354.
Y. Liu et al. / International Journal of Pharmaceutics 476 (2014) 169–177 Sek, L., Porter, C.J., Charman, W.N., 2001. Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products by HPTLC coupled with in situ densitometric analysis. Biomed. Anal. 25, 651–661. Shangguan, M., Lu, Y., Qi, J., Han, J., Tian, Z., Xie, Y., Hu, F., Yuan, H., Wu, W., 2014. Binary lipids-based nanostructured lipid carriers for improved oral bioavailability of silymarin. J. Biomater. Appl. 28, 887–896. Thomas, N., Holm, R., Mullertz, A., Rades, T., 2012. In vitro and in vivo performance of novel supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS). J. Control. Release 160, 25–32.
177
Tian, Z.Q., Qi, J.P., i, Y., Han, J., Yuan H. l. Zhang, X., Xie, Y.C., Hu, F.Q., Lu, Y., Wu, W., 2013. Solidification of nanostructured lipid carrier (NLC) onto pellets by fluidbed coating: preparation, in vitro characterization and bioavailability in dogs. Powder Technol. 247, 120–127. Williams, H.D., Trevaskis, N.L., Yeap, Y.Y., Anby, M.U., Pouton, C.W., Porter, C.J., 2013. Lipid-based formulations and drug supersaturation: harnessing the unique benefits of the lipid digestion/absorption pathway. Pharm. Res. 30, 2976–2992. Wu, W., Wang, Y., Que, L., 2006. Enhanced bioavailability of silymarin by selfmicroemulsifying drug delivery system. Eur. J. Pharm. Biopharm. 63, 288–294.