Oral bioavailability of silymarin formulated as a novel 3-day delivery system based on porous silica nanoparticles

Acta Biomaterialia 8 (2012) 2104–2112

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Oral bioavailability of silymarin formulated as a novel 3-day delivery system based on porous silica nanoparticles Xia Cao 1, Min Fu 1, Liang Wang 1, Hongfei Liu 1, Wenwen Deng, Rui Qu, Weiyan Su, Yawei Wei, Ximing Xu ⇑, Jiangnan Yu ⇑ Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District, Zhenjiang 212001, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 17 October 2011 Received in revised form 4 February 2012 Accepted 9 February 2012 Available online 17 February 2012 Keywords: Porous silica nanoparticles Controlled release Bioavailability Poorly soluble drug Silymarin

a b s t r a c t The purpose of this study was to develop porous silica nanoparticles (PSNs) as a carrier to improve oral bioavailability of poorly water-soluble drugs, using silymarin as a model. PSNs were synthesized by reverse microemulsion and ultrasonic corrosion methods. A 3-day release formulation consisting of a silymarin solid dispersion, a hydrophilic gel matrix and silymarin-loaded PSNs was prepared. In vitro release studies indicated that both the silymarin-loaded PSNs and the 3-day release formulation showed a typical sustained-release pattern over a long period, about 72 h. The in vivo studies revealed that the 3-day release formulation gave a significantly higher plasma concentration and larger area under the concentration–time curves than commercial tablets when orally administered to beagle dogs. This implies that the prepared 3-day release formulation significantly enhanced the oral bioavailability of silymarin, suggesting that PSNs can be used as promising drug carriers for oral sustained release systems. Thus providing a technically feasible approach for improving the oral bioavailability and long-term efficacy of poorly soluble drugs. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Silymarin, an antihepatotoxic polyphenolic substance extracted from fruit seeds of the milk thistle plant (Silybum marianum Gaertn), has been widely used to treat a variety of liver disorders including acute and chronic viral hepatitis [1–3], alcoholic liver diseases [1], toxin- and drug-induced hepatitis and cirrhosis, fatty liver radiation, and toxicity. It is mainly composed of four flavonolignans, silybin, isosilybin, silydianin and silychristin, of which silybin is the most biologically active component, representing approximately 60–70% [3–5]. However, the therapeutic effects of silymarin are restricted due to its poor water solubility, resulting in poor oral absorption and low bioavailability after oral administration [6–8]. Xiao and co-workers reported that after oral administration of pure silybin, it was not detected in plasma [9]. In addition, Wu and co-workers reported that absolute oral bioavailability of silybin in rats was approximately 0.95% due to the poor solubility and extensive pre-systemic metabolism of the drug [10]. It was also reported that only 20–50% of silymarin was absorbed from the gastrointestinal tract after oral administration [6]. Furthermore, silymarin’s poor water solubility and poor ⇑ Corresponding authors. Tel./fax: +86 511 85038451. 1

E-mail addresses: [email protected] (X. Xu), [email protected] (J. Yu). These authors contributed equally to this work.

absorption may be attributed to its poor permeation across intestinal epithelial cells [11,12]. Therefore, developing strategies to overcome these difficulties and to enhance the oral bioavailability of silymarin are highly desirable. In recent times, different strategies have been investigated to improve the dissolution and bioavailability of silymarin. These strategies included silymarin/polyvinylpyrrolidone solid dispersion (SD) pellets [13], a silymarin or dehydrosilymarin proliposome [9,14,15], a silymarin self-microemulsifying drug delivery system (SMEDDS) [16], a silybin–phospholipid complex [17], silybinloaded povidone–sodium cholate–phospholipid mixed micelles [18] and silymarin liposomes [19]. Again, Xiao and co-workers reported that a silymarin proliposome improved the oral bioavailability of silymarin in beagle dogs and enhanced gastrointestinal absorption. The relative bioavailability of silymarin SMEDDS is superior to that of silymarin PEG 400 solution and its suspension. Although the oral administration of these drug delivery systems in previous studies showed improved bioavailability, only few of these formulations that displayed sustained release of silymarin for more than 16 h in vivo have been reported. Thus improvement in bioavailability has been limited. Over the past few decades, the application of mesoporous silica nanoparticles (MSNs) as a drug delivery system has attracted considerable attention [20–22]. It was reported that ordered mesoporous silica materials could be developed into a broad-spectrum

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.02.011

X. Cao et al. / Acta Biomaterialia 8 (2012) 2104–2112

formulation platform for poorly soluble drugs [20]. MSNs possess many unique features such as excellent biocompatibility and biodegradability, a large specific surface area and pore volume, tunable pore size with a narrow pore size distribution and excellent physicochemical stability. The porous structures with hundreds of empty channels (mesopores) which can also be modified easily are able to absorb/encapsulate relatively large amounts of drug molecules, thus allowing the control of drug release with high precision. Ukmar and co-workers found out that the controlled release of drugs from ordered porous materials had a close relationship with the pore size and drug molecule–wall attractions [23–25]. Drug–wall interactions can be achieved via the surface modification of the drug-delivery materials [23,26]. A number of studies have reported the sustained release of drug-loaded porous silica nanoparticles (PSNs) in vitro. Li and co-workers reported the typical sustained release of Brilliant Blue F-loaded porous hollow silica nanoparticles (PHSNPs) in vitro, from which drug dissolution was observed for as long as 1140 min [27]. In addition, Chen and coworkers found out that the release of cefradine from PHSNP followed a three-stage pattern lasting for 12 h [28] and was due to the drug release from the surface pore channels in the wall and the inner hollow part of PHSNPs. Safety has also been investigated with an in vivo study showing that MSNs exhibited no visible cytotoxicity against LX-2 cells over a broad spectrum of concentrations and possessed good blood compatibility [29]. Furthermore, Bimbo and co-workers reported that 80% of PSNs were dissolved in biorelevant media after 144 h [30]. They also found that the degradable product was harmless to Caco-2 human epithelial colorectal adenocarcinoma cells [31]. In all, the PSNs exhibited excellent biocompatibility, biodegradation, in vivo stability, low cytotoxicity and nonimmunogenic profiles [31], thereby making these nanoparticles an ideal candidate for oral drug delivery. However, very few studies have focused on the in vivo pharmacokinetics and bioavailability of drug-loaded PSNs, which is of great importance for the evaluation of in vitro and in vivo correlations. It is the aim of this study to develop an approach for in vivo evaluation of drug-loaded PSNs for better investigation of the efficacy of prepared formulations. Improving the solubility of poorly soluble drugs and preparing sustained-release formulations have always been a great challenge to researchers. Poorly soluble drugs are solubilized prior to being developed into sustained-release formulations that can typically release for 12 h or at most 24 h, with one or two administrations per day. With regard to the promising properties of PSNs, this study was targeted at developing PSNs as carriers of the poorly soluble drug silymarin in order to improve its bioavailability. This obviously provided the basis for the development of a new longacting sustained-release formulation for poorly soluble drugs. The study employed a reverse microemulsion method for the synthesis of monodispersed nonporous silica nanoparticles to form ‘‘cores’’ and an ultrasonic corrosion method to create regular nanometer-sized pores with sodium carbonate (Na2CO3) solution. The pharmacokinetics of drug-loaded PSNs and their formulation was investigated in beagle dogs and the oral bioavailability was compared with that of LegalonÒ, a commercial product.

2. Materials and methods 2.1. Materials Silymarin was kindly supplied by Jiangsu Zhongxing Pharmaceutical Co., Ltd. (Zhenjiang, PR China). Commercial silymarin tablets (LegalonÒ, MADAUS GmbH, Germany) were used as a reference product. Tetraethoxysilane (TEOS), hydroxypropyl methylcellulose (HPMC K4M) and low-substituted hydroxypropyl cellulose (L-HPC)

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were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, PR China). Lecithin (Leci) and Eudragit E100 were purchased from Shanghai Chineway Pharmaceutical Technical Company, Ltd. (Shanghai, PR China). Octylphenol polyoxyethylene (NP-10) was provided by Shanghai Junhui Chemical Factory (Shanghai, PR China), cyclohexane (CHX) by Shanghai Chemical Reagent (Shanghai, PR China), a-naphthol by Tingxin Chemical Reagent Factory (Shanghai, PR China), polyvinylpyrrolidone K30 (PVP K30) by Shanghai Sunpower Chemical Co., Ltd. (Shanghai, PR China), Na2CO3 by Shanghai Hongguang Chemical Factory (Shanghai, PR China) and dialysis bags by Shanghai Yuanju Biologic Technology Co., Ltd. (Shanghai, PR China). All other chemicals and solvents were of analytical grade. Double-distilled water was freshly prepared in the laboratory. 2.2. Synthesis of solid nanoparticles (SNs) The SNs were synthesized by the reverse microemulsion method. NP-10, butanol and 2 ml ammonia (25.6%, w/v) were mixed with 50 ml CHX, and agitated for 15 min to obtain the reversed emulsion. The TEOS was slowly added to the system with stirring for 24 h. An equal volume of alcohol was also added to the emulsion, which was then sonicated at 300 W for 30 min. The resulting nanoparticle suspension was centrifuged at 15000 g for 15 min at 4 °C using an ultracentrifuge (Heraeus Biofuge, Stratos, Germany). The pellet was washed three times with ethanol followed by double-distilled water successively. Finally, a small amount of doubledistilled water was added to the resulting product, which was then lyophilized to obtain SNs. 2.3. Synthesis of porous silica nanoarticles The PSNs were prepared by the ultrasonic corrosion method. The SNs (100 mg) were added to 100 ml of 0.6 M Na2CO3 solution and the resulting suspension sonicated at 200 W at 65 °C. It was then centrifuged at 15,000 g for 15 min at 4 °C. The precipitate was washed with double-distilled water several times and lyophilized to obtain PSNs. According to the previous studies, temperature (A), ultrasonic time (B) and ultrasonic power (C) had the most significant effects on the properties of prepared PSNs [32]. The optimum preparation conditions were obtained by orthogonal experimental design and single factor experimentation, thus arranging the three factors above with four levels for each factor. Sixteen different sets of experiments were performed under conditions of different parameter combinations according to the standard L16 (45) table as shown in Table 1. Table 2 shows the variance analysis of three factors on PSNs. 2.4. Drug impregnation in PSNs and SNs The PSNs (2 g) were immersed in an ethanol solution of silymarin at a concentration of 0.2 g ml1 and stirred for 24 h. The resulting suspension was purified by centrifugation. After the removal of the supernatants, 20 ml ethanol was added to the nanoparticles and the solution was thoroughly mixed on a vortex mixer for 1 min. It was then centrifuged at 10,000 g for 5 min with a Mini Sin centrifuge (Eppendorf, Germany). This step was repeated three times to remove silymarin adsorbed on the surface of PSNs. The silymarin concentration in the ethanol solution was then determined with a UV-Vis spectrophotometer (UV-2401PC, Shimadzu). The resulting product was resuspended in doubledistilled water and freeze-dried to obtain silymarin-loaded PSNs. SNs were also taken through the same procedure to obtain silymarin-loaded SNs.

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Table 1 Orthogonal design table L16 (45) of PSNs. Facter

(A)

(B)

(C)

Blank 1

Blank 2

Result

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean 1 Mean 2 Mean 3 Mean 4 Range

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 47.375 64.750 77.250 66.875 29.875

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 64.375 63.750 63.500 64.625 1.125

1 2 3 4 2 1 4 3 3 4 1 2 4 3 2 1 53.875 60.375 66.875 75.125 21.250

1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3 62.625 62.625 65.125 66.000 3.500

1 2 3 4 4 3 2 1 2 1 4 3 3 4 1 2 63.500 64.750 62.875 65.125 2.250

35.5 42.5 49.5 62 63.5 55 74.5 66 83 88.5 66 71.5 75.5 69 64 59 / / / / /

Table 2 Variance analysis of three factors on PSNs. Factor

Residual sum of squares

Degree of freedom

F ratio

F critical value

Significance level

Temperature

1843.063

3

72.455

19.000

Ultrasonic time Ultrasonic power

3.313

3

0.130

19.000

Highly significant (p < 0.01) Not significant

990.688

3

38.946

19.000

38.222

6

/

/

Error

Highly significant (p < 0.01) /

Similarly, silymarin SD, silymarin-loaded PSNs, HPMC K4M and L-HPC of given quantities as listed in Table 3 were uniformly mixed, and the procedure was repeated as above to obtain granule B. The mixture of granule A and granule B was filled into capsules to obtain a 3-day release formulation. 2.6. Characterization of PSNs The internal porous structures of PSNs and silymarin-loaded PSNs were observed by transmission electron microscopy (TEM; JEM-2100, JEOL, Japan). The particle size and polydispersity of the PSNs and SNs were determined using a particle size analyzer (BI90Plus, Brookhaven Instruments Corporation, Holtsville, USA). Measurements were performed in triplicate following a 1/100 (v/v) dilution of nanoparticle suspension in double-distilled water at 25 °C. The polydispersity index range was between 0 and 1. Additionally, the hydrodynamic particle size of silymarin-loaded PSNs was determined by photon correlation spectroscopy (PCS) on an ALV 5000 (Laser Vertriebsgesellschaft GmbH, Langen, Germany) at a scattering angle of 90° (sampling time: 200 s). The specific surface area, pore volume and pore diameter of the samples were measured by the nitrogen (N2) adsorption method using a high-speed automated surface area and pore size analyzer (NOVA 2000, Quantachrome, USA) according to the Brunauer–Emmett–Teller and Barrett–Joyner–Halenda procedures. Powder X-ray diffraction (PXRD) analysis was carried out using an X-ray diffractometer (D/max A type, Rigaku Denki, Japan) using Cu Ka (40 kV, 20 mA) radiation. The degree of diffraction was measured at 5° min1 every 0.01° between 5° and 45° (2h). Differential scanning calorimetry (DSC) was then performed using a differential scanning calorimeter (DSC-PYRIS-1, Phillips, the Netherlands) to study the interactions between the drug molecules and the channel walls. The samples were heated in hermetically sealed aluminum pans at a scanning rate of 5 °C min-1 from 10 ± 0.2 to 530 ± 0.2 °C. An empty aluminum pan was used as a control. 2.7. In vivo studies

Table 3 Constituents of sustained-release granules in the 3-day release formulation.

Silymarin SD Silymarin-loaded PSNs HPMC K4M L-HPC

Granule A

Granule B

175 / 35 40

117 92 37.5 37.5

2.5. Preparation of a 3-day release formulation Silymarin solid dispersion (SD) was prepared by the solvent evaporation method. After 100 mg silymarin, 200 mg PVP K30, 50 mg Leci and 30 mg Eudragit E100 were dissolved in absolute ethyl alcohol, the solvent was removed under reduced pressure using a rotary evaporator at 50 °C, 90 rpm and then dried on a water bath at 60 °C. The sample was kept at 20 °C for 2 h to solidify and then dried in an oven at 60 °C for 24 h. The dried sample was pulverized using a mortar and pestle. It was then sieved through a 210 lm sieve (mesh size, 80), and then stored in a dessicator at room temperature to obtain silymarin SD. Sustained-release granules were prepared by the wet granulation method. Table 3 lists the constituents of the sustained-release granules. A 70% syrup was added to a mixture of silymarin SD, HPMC K4M and L-HPC (see Table 3) to prepare a soft material. The resultant soft material was granulated with a 16 mesh sieve and dried at 60 °C for 30 min. It was then sieved through another 16 mesh sieve to obtain Granule A.

2.7.1. Animal samples Healthy male beagle dogs of an average weight of 10 ± 2 kg were fasted overnight before the drug administration but allowed free access to water throughout the study. The drug was administered as an oral dose (21.2 mg kg1) to four groups of dogs (6 dogs per group). The first group received commercial tablets, the second group received gelatin capsules filled with silymarin SD, the third group was treated with gelatin capsules filled with silymarinloaded PSNs, and the fourth group received 3-day release formulations. Serial blood samples (200 ll) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 60, 72 h after oral administration. The plasma obtained after centrifugation (10 min, 4000 rpm) was immediately stored at 20 °C until further analysis. After thawing, 200 ll of plasma was mixed with 100 ll of phosphate buffer (pH 5.0). The mixture was vortexed and incubated for 16 h at 37 °C. The liquid–liquid extraction was then accomplished by adding 20 ll of internal standard solution (10 lg ml1 in methanol) followed by a gentle vortex agitation (5 min). The resulting mixture was centrifuged for 10 min at 4000 rpm and the organic layer was transferred to a clean vial. This was then evaporated under N2 at 30 °C. The residue was dissolved in 100 ll of mobile phase and analyzed using high-performance liquid chromatography (HPLC). 2.7.2. HPLC analysis Since silybin is the main biologically active component of silymarin, the pharmacokinetics and bioavailability of silymarin are usually characterized by the determination of plasma silybin

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concentration [12,32,16]. Plasma silybin concentration was determined by HPLC. A Nova-Pak C18 column (150 mm  3.9 mm, 5 lm) with a column temperature at 37.5 °C and a mobile phase consisting of methanol and KH2PO4 (0.05 mol l1) with a ratio of 9.9:11 at pH 3.8 were used. The eluent was monitored at 288 nm with a flow rate of 1 ml min1, and a-naphthol was used as an internal standard. 2.7.3. Analysis of pharmacokinetic parameters The pharmacokinetic parameters of the drug characterized by the peak concentration in plasma (Cmax), the time to attain peak concentration (Tmax), the mean residence time (MRT) and the area under the concentration–time curve (AUC) were derived by the pharmacokinetic software BAPP2.3 (supplied by the Center of Drug Metabolism, China Pharmaceutical University, PR China). The relative bioavailability (F) of the drug was determined by calculating the ratio of AUC for the test formulation (AUCT) and AUC for the reference formulation (AUCR) according to the following equation:

Fð%Þ ¼

AUC T  DR  100%; AUC R  DT

ð1Þ

where DT and DR represent the oral dose of the test and reference formulations, respectively. 2.8. In vitro release studies Silymarin-loaded PSNs, SNs and the 3-day release formulation were used for the in vitro release studies. The amount of silymarin released from each sample was determined by the dialysis tube method in a dissolution test apparatus (Tianjin University Radio Factory, Tianjin, PR China) using the paddle method (Chinese Pharmacopoeia 2005). The paddles were rotated at 100 rpm for 72 h. An accurately weighed quantity of each sample was put into the dialysis bags. The bags were then fixed to the paddles and placed in a dissolution medium (900 ml artificial intestinal or gastric juice, adjusted temperature of 37 ± 0.1 °C). The artificial intestinal juice was prepared by dissolving potassium dihydrogen phosphate in double-distilled water and adjusting the pH of the solution with 1 M sodium hydroxide solution to 6.8. This was mixed with an aqueous solution containing 10 g of pancreatin and diluted up to 1000 ml with double distilled water. The artificial gastric juice (pH 1.2) was also prepared by adding 800 ml double-distilled water and 10 g pepsin to 16.4 ml diluted HCl. The solution was then diluted with double-distilled water up to 1000 ml. Test fluids were withdrawn at predetermined time intervals from each vessel and were then filtered through a 0.45 lm membrane filter. The same volume of fresh medium was used to replace it. The quantity of silymarin in the filtrate (20 ll) was analyzed using an HPLC system (Waters, Model 510) equipped with an UV detector (Waters, Model 486). The HPLC conditions were: reverse-phase column, Nova-Pak C18 column (3.9  150 mm, 5 lm) at 37 °C; mobile phase, dipotassium hydrogen phosphate (0.05 M):methanol = 9.9:11 (v/v) mixture; flow rate, 1.0 ml min1; and detection wavelength, 288 nm. All experiments were performed at 37 °C and at least in triplicate. The standard curve range was 1.68–31.8 lg ml1. 3. Results and discussion 3.1. Characterization of PSNs The silymarin concentration was found to be 68.52% in PSNs, much more than the 5.64% found in SNs: the PSNs could carry greater amounts of drugs due to their structure being more porous than that of the SNs. Additionally, the drug-loading rate and the

Fig. 1. TEM photographs of (a) PSNs, scale bar = 50 nm and (b) silymarin-loaded PSNs, scale bar = 100 nm.

molecular organization of the poorly water-soluble drugs in the pores can be affected by the co-adsorbed water inside the porous materials [33]. The TEM image of PSNs showed a rough surface and porous structures (Fig. 1a), while silymarin-loaded PSNs possessed a smooth surface and few porous structures (Fig. 1b). The TEM images also revealed the particle sizes of PSNs and drugloaded PSNs, which range from 50 to 60 nm. As shown in Fig. 2a, the particle size distributions of SNs and PSNs were narrow, with the average particle size of SNs being approximately 60 nm, while that of PSNs was slightly smaller (56.3 nm). This could possibly be due to the fact that part of the outer surface of the silica nanoparticle was dissolved during the process of producing PSNs by the ultrasonic corrosion method. The result of the PCS showed that the mean hydrodynamic particle diameter of silymarin-loaded PSNs was 56.2 nm (PDI: 0.248) (Fig. 2b), which revealed a unimodal and narrow size distribution. This was in agreement with the TEM images (Fig. 1). As shown in Fig. 3, PSNs exhibited an adsorption isotherm similar to type II adsorption isotherms and a hysteresis loop in the range of relative pressure from 0.6 to 0.9. This indicated the presence of mesopores in PSNs [34]. The pore size distribution of PSNs was narrow with pore diameter between 5 and 15 nm. However, the hysteresis loop was not observed for SNs (Fig. 4), which indicated the absence of pores. When loaded with the drug, the PSNs revealed a narrow hysteresis loop, suggesting a limited size and number of pores (Fig. 5). This could be due to the drug loading.

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Fig. 4. N2 adsorption–desorption isotherms of SNs.

Fig. 2. Particle size distribution of PSNs.

Fig. 5. N2 adsorption–desorption isotherms of silymarin-loaded PSNs.

Fig. 3. N2 adsorption–desorption isotherms of PSNs.

N2 adsorption results indicated that PSNs have a larger specific surface area (249.95 m2 g1) than SNs (67.91 m2 g1), and the pore volumes of PSNs and SNs were 0.39 and 0.13 cm3 g1, respectively. These results may also be due to the porosity of PSNs. PXRD patterns of silymarin, PSNs and silymarin-loaded PSNs are shown in Fig. 6. Pure silymarin powder showed numerous distinctive peaks. The PXRD pattern of PSNs was amorphous with no diffraction peaks. For silymarin-loaded PSNs, diffraction peaks derived from pure silymarin disappeared. The absence of drug peaks suggested that the silymarin was encapsulated in the pores of PSNs and transformed to an amorphous state [35,22]. The DSC thermograms of silymarin, PSNs and silymarin-loaded PSNs are shown in Fig. 7. In the case of silymarin, two obvious exothermic peaks were observed at 289.9 and 428.5 °C. No exothermic peak appeared in the case of PSNs. However, when loaded into PSNs, the two exothermic peaks of silymarin disappeared, and a

Fig. 6. PXRD patterns of silymarin, PSNs and silymarin-loaded PSNs.

very wide peak appeared at 340 °C. This means that silymarin was adsorbed inside the PSNs channels, and there was an attraction between silymarin and channel walls. 3.2. In vivo pharmacokinetics The profiles of the plasma silybin concentration attained by the 3-day release formulation with different ratios of SD in Granule A,

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Fig. 7. Differential scanning calorimetry (DSC) thermograms of silymarin, PSNs and silymarin-loaded PSNs. Fig. 9. Mean plasma concentration–time curves of silybin after oral administration of commercial tablets, silymarin SD, silymarin-loaded PSNs, and 3-day release formulation to beagle dogs. Data are expressed as the mean ± SD (n = 6).

Fig. 8. Mean plasma concentration–time curves of 3-day release formulation with different ratios of SD in Granule A, SD in Granule B, and drug-loaded PSNs.

SD in Granule B and drug-loaded PSNs are shown in Fig. 8. When SD in Granule A, SD in Granule B and drug-loaded PSNs were formulated at the ratios of 3:3:6 and 3:3:4, respectively, the profiles of plasma silybin concentration in both cases were found to be divided into two maximum concentration peaks, indicating an undesirable fluctuation. In the case of 3:2:3, a stable plasma silybin concentration was maintained. This implies that the formulated ratio had a greater effect on the release behavior of the drug after oral administration to the beagle dogs. As a result, the 3-day release formulation with the ratio of 3:2:3 (SD in Granule A, SD in Granule B and drug-loaded PSNs) was used to compare with the commercial tablets, silymarin SD and silymarin-loaded PSNs in the in vivo and in vitro release experiments. Mean plasma concentration–time curves of silybin after oral administration of commercial tablets, silymarin SD, silymarinloaded PSNs, and 3-day release formulation to beagle dogs at 21.2 mg kg1 are shown in Fig. 9. The dosage was referred to as the recommended dose of the present commercial preparation. Silica is classified as a non-hazardous and non-restrictive substance. The FDA approves a maximum of 2 wt.% silica for food use, and for pharmaceutical use up to 3 wt.% in internal applications and up to 8 wt.% in topical applications [36]. In this study, the amount of silica nanoparticles used in oral administration in a single dose was 1.59 wt.%, lower than the maximum amount of silica for food use (2 wt.%). This indicated that the silica nanoparticles used in this study were non-toxic.

With respect to the commercial tablets, the plasma silybin concentration appeared to increase progressively and Cmax (490.9 ng ml1) was reached with Tmax at approximately 1 h, followed by a rapid decline and elimination of silybin from Tmax until 72 h after administration. A double peak of silybin maximum concentration was observed for the commercial tablets, which was considered to be characteristic of enterohepatic circulation [16,37]. Silymarin SD gave the lowest Cmax (236.7 ng ml1) at approximately 3 h, which was approximately 2-fold lower than the commercial tablets. The plasma silybin concentration of the silymarin SD was found to be below the detection limit of HPLC at 24 h after oral administration. Interestingly, when silymarin-loaded PSNs were orally administrated to beagle dogs, the plasma silybin concentration was characterized by a delayed release, with the silybin concentration lower than that of the commercial tablets and silymarin SD until 5 h after administration. A similar delayed-release effect had also been reported by Chen and co-workers in their in vitro release study of drug-loaded PHSNPs [28]. However, the concentration of silybin in plasma drastically increased from approximately 16 h and Cmax of silymarin-loaded PSNs (241.2 ng ml1) was seen at 24 h after administration. Moreover, the high plasma concentration was maintained even up to 72 h. This indicated that silymarin-loaded PSNs may have caused a delay in the rate of absorption of the drug, but enhanced the extent of absorption as evident in the significant difference between the AUCs of commercial tablets and silymarinloaded PSNs. Another observation was that silymarin-loaded PSNs exhibited a multistage release pattern during the first 24 h. Such release features in in vivo studies have also been reported to be characteristic of PSNs [29]. In a related study, the silymarin was encapsulated in the pore channels or the internal core of PSNs in the amorphous state. The release of the encapsulated silymarin depended mainly on molecular diffusion through the pore channels, thereby producing a favorable controlled release [22]. These results also suggested that the prepared PSNs could be used as a promising drug carrier for oral sustained-release systems, satisfying the need for prolonged treatment after administration. As shown in Fig. 9, the 3-day release formulation, consisting of silymarin SD, HPMC K4M, L-HPC and silymarin-loaded PSNs with the optimal ratio as mentioned above, showed a rapidly increased plasma concentration after administration. Afterwards, a sustained release with a high plasma concentration was found to be maintained throughout the experiment up to 72 h. Cmax and Tmax of

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Table 4 Main pharmacokinetic parameters in humans, beagle dogs and rabbits.

Human Rats Beagles Rabbits Beagles Beagles

Formulation

Dosage (mg/kg)

Cmax (ng/ml)

Tmax (h)

t1/2 (h)

AUC0-1 (ng h/ml)

Silybin–phosphatidylcholine complex Silybin–phospholipids complex [17] Silymarin proliposome [9] Silymarin SMEDDS [16] Silymarin-loaded PSNs 3-Day release formulation

7.2 9.2 7.7 300 21.2 21.2

298 ± 96 126.72 472.62 ± 126.91 1010 ± 210 241.2 338.1

1.6 ± 0.3 0.17 0.5 ± 0.21 4.33 ± 0.82 24 10

/ / 1.61 ± 0.46 6.53 ± 1.02 22.3 54.3

881 ± 207 1020.33 2464.62 ± 579.35 6230 ± 1750 14330.1 25819.4

the 3-day release formulation were 338.12 ng ml1 and 10 h, respectively. All of these findings indicated that the 3-day release formulation enhanced both the absorption rate and bioavailability of the drug. The initial quick release and significantly improved absorption of the drug are attributed to the presence of silymarin SD in the 3-day release formulation, which allows the relatively rapid release of the drug to cover the delayed-release effect caused by PSNs. The sustained characteristic is also attributed to silymarin-loaded PSNs due to the controlled release from the pore channels of loaded PSNs. The addition of the common slow-release material HPMC K4M significantly contributed to elimination of the obvious peak–valley phenomenon observed in the dissolution of the preparation consisting of only silymarin SD and silymarinloaded PSNs in the previous study [18]. The transition effect of HPMC K4M gave rise to stable and continual release for 3 days. The in vivo transport mechanism of PSNs is under study and an explanation for the prolonged absorption will be given in future works. Although PSNs have been reported to possess slow avermectin release for nearly 30 days [38], the instability of the released drug in the medium should be considered. Moreover, it is not good for an orally administered preparation to have such a long administration period if the bioavailability and pharmacological actions have not been proven to be desirable. The pharmacokinetic parameters for the different formulations are summarized in Table 4. Remarkably, the 3-day release formulation showed an outstanding sustained-release property after oral administration to beagle dogs, for which the elimination half-life (t1/2) was 54.3 h, approximately 2.43- and 5.72-fold higher than that of silymarin-loaded PSNs and commercial tablets, respectively. Furthermore, MRT of the 3-day release formulation was 70.2 h, significantly higher than that of silymarin-loaded PSNs and commercial tablets. More importantly, the AUC0-1 of commercial tablets drastically increased from approximately 3.56 lg h ml1 to approximately 14.3 and 25.8 lg h ml1 for drug-loaded PSNs and the 3-day release formulation, respectively. The relative oral bioavailability of the 3-day release formulation was 448.2% and that of silymarin-loaded PSNs was approximately 323.9%. Although the relative bioavailability of silymarin SD was found to be only 29.6%, it contributed to the relatively fast drug release in the initial phase. The combination of silymarin SD and silymarin-loaded PSNs that made up the 3-day release formulation produced relatively high long-term release rates and concentrations, which resulted in the most favorable bioavailability. The oral bioavailability of silymarin has been reported previously to be poor or non-existent [9,10]. In this study, the prepared PSNs formulated as a 3-day release preparation considerably improved the oral bioavailability of silymarin. This could probably be due to at least three different mechanisms or a combination of them. Firstly, the prepared PSNs may have improved the solubility of silymarin in the gastrointestinal tract by attainment of high local concentrations and even drug supersaturation, thereby effectively enhancing the drug absorption [39,40]. Another explanation could be that PSNs effectively enhanced the permeation of the drug across the monolayer of the intestinal epithelial cell, which is the main rate-limiting barrier for drug absorption/diffusion. These probable mechanisms had been proven by Bimbo and co-workers

[30], who demonstrated that MSNs increased the permeation of a poorly soluble model drug across the monolayers of differentiated Caco-2 cells. Lastly, improved oral bioavailability could be attributed to the specific long-term release pattern of silymarin from PSNs, lasting for approximately 72 h with high plasma concentrations. Hence, many different alternative approaches to enhancing the oral bioavailability of silymarin had been reported by other researchers including SMEDDS [16], a silybin–phospholipid complex [17], a silybin–phosphatidylcholine complex [11], liposomes [19] and proliposomes [9]. The main pharmacokinetic parameters are compared in Table 4, which shows that silymarin-loaded PSNs and the 3-day release formulation exhibited significantly greater relative bioavailability and more favorable sustained-release properties than those previously investigated. 3.3. In vitro release studies Fig. 10 shows the in vitro release profiles of silymarin-loaded SNs, PSNs and 3-day release formulation in both artificial gastric and intestinal juices, respectively. In artificial gastric juice, SNs rapidly achieved an accumulated silymarin release of 78.5% in 0.5 h and reached 84.9% at 4 h after oral administration, while in artificial intestinal juice, SNs achieved an accumulated release of 99.5% in 2 h and finally 99.7% at 4 h after oral administration (Fig. 10a). This was probably due to the lack of storage capacity for SNs with non-porous structures. When only loaded on the surface of SNs, silymarin possessed a burst release and almost complete release after 4 h of oral administration, showing no sustained release property. However, in the case of PSNs, a sustained release for 72 h was accomplished (Fig. 10b). The amount of dissolved silymarin in 72 h accumulated to 13.3% in artificial gastric juice, which was higher than 8.2% in artificial intestinal juice. According to the research report by Li and co-workers, the in vitro release rate was controlled by diffusion of drug molecules through the pores of PSNs [27]. The small fractions of released drug from PSNs in artificial gastric and intestinal juices were indicative of attractive interactions between the drug and the silica walls. As mentioned earlier, the in vitro release behavior of the porous silica materials was associated with the pore size and drug–matrix interactions. Horcajada and co-workers found that in a simulated body fluid solution the drug delivery rate from mesoporous materials decreased as the pore size decreased in the range of 3.6–2.5 nm [24]. Furthermore, it had been reported that the release curves from mesoporous materials are significantly affected by drug–matrix interactions which affect the relative cross-section of pores where the local flux has a non-vanishing axial component and in turn control the effective transfer of drug into bulk solution [23,25]. Future investigation in this direction will be needed to clarify this issue. The in vitro dissolution profiles of the 3-day release formulation are shown in Fig. 10c. The dissolution profiles of the 3-day release formulation in artificial gastric and intestinal juices were similar, with the accumulated release rates in both cases being lower than 15% up to 72 h. In comparison with the SNs, the 3-day release formulation showed a significant sustained-release effect. This could

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Fig. 10. In vitro release profiles in artificial gastric or intestinal juice of (a) silymarin-loaded SNs, (b) silymarin-loaded PSNs and (c) 3-day release formulation.

Table 5 Drug-release models of silymarin-loaded PSNs and 3-day release formulation. Formulations

Artificial gastric juice

silymarin-loaded PSNs

¼ 0:0017t þ 0:0287 ðR2 ¼ 0:8989Þ a
Mt ¼ 0:0019t  0:0288 ðR2 ¼ 0:9106Þ In 1  M1 Mt M1

3-day release formulation

a

c

¼ 0:0161t 12 þ 0:0034 ðR2 ¼ 0:9745Þ

c

¼ 0:001t þ 0:0206 ðR2 ¼ 0:7233Þ a
Mt ¼ 0:0011t  0:0209 ðR2 ¼ 0:7318Þ In 1  M1 ¼ 0:0103t 12 þ 0:0049 ðR2 ¼ 0:894Þ

c

¼ 0:0007t þ 0:0073 ðR2 ¼ 0:8609Þ a
Mt ¼ 0:0007t  0:0073 ðR2 ¼ 0:8646Þ In 1  M1 Mt M1

b

Mt M1

Mt M1

Mt M1

b

Artificial intestinal juice

Mt M1

¼ 0:0063t 12  0:0028 ðR2 ¼ 0:9677Þ

b

c

¼ 0:0018t þ 0:0162 ðR2 ¼ 0:8735Þ a
Mt ¼ 0:002t  0:0161 ðR2 ¼ 0:8854Þ In 1  M1 Mt M1

b

Mt M1

¼ 0:0173t 12  0:0087 ðR2 ¼ 0:9692Þ

b

c

Zero-order kinetics. First-order kinetics. Higuchi equation.

probably be due to the ‘‘triple release’’ mechanism consisting of quick release of the solid dispersion, regular slow release of the hydrophilic gel matrix and the long-acting slow release of the PSNs. In particular, the regular slow release of the hydrophilic gel matrix which maintained the effective plasma concentration for about 24 h. The in vitro drug release models of silymarin-loaded PSNs and 3-day release formulation are presented in Table 5. Three different models (zero-order kinetics, first-order kinetics and the Higuchi equation) were employed to fit the release profiles of silymarin-loaded PSNs and 3-day release formulation in artificial gastric and intestinal juices. Interestingly, the in vitro release profiles of silymarin-loaded PSNs and 3-day release formulation in both artificial gastric and intestinal juices can be best fitted to the Higuchi equation. The Higuchi equation is a typical sustained-release model, indicating the sustained-release effect of PSNs and 3-day release formulation. The diffusion mechanism of drug release from the pores of PSNs was well described by Zhu

et al. [27]. The release mechanism here is drug diffusion from the PSNs and matrix pores, and the diffusion rate from the pores is the rate-limiting step of the drug dissolution. Although silymarin-loaded PSNs exhibited poor dissolution of silymarin in such regular dissolution media, it can be inferred that the in vitro release profiles may not be associated with in vivo pharmacokinetics. It had been reported by other researchers that there are many factors that influence the release of drug-loaded nanoparticles in vitro: the size of the nanoparticles, the percentage of drug-loaded particles and pH [22], temperature [22] and the concentration of dissolution medium [23]. In this study, measurements such as adjusting pH and adding surfactant were taken into consideration to achieve a desirable in vivo and in vitro correlation, but no acceptable results were obtained. An appropriate dissolution medium will be selected in the next study to achieve a more reliable assessment of silymarin-loaded PSN release in vitro, which would be in agreement with the plasma profiles.

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4. Conclusion A novel approach has been developed in preparing PSNs for controlled release of a poorly soluble drug, silymarin, in order to improve its bioavailability. The prepared PSNs exhibited a large specific surface area and narrow pore size distribution of approximately 10 nm. Silymarin-loaded PSNs showed a typical sustainedrelease pattern of approximately 72 h in vivo. The prepared 3-day release formulation gave an initial burst release from SD, and a sustained release due to the PSNs. The smooth and stable connection of the two actions were attributed to the hydrophilic gel matrix, which showed a considerable increase in bioavailability compared to commercial tablets. Overall, the study provided an approach to improve the oral bioavailability and long-term efficacy of silymarin via the use of PSNs, indicating a new strategy for developing sustained-release formulations of poorly soluble drugs. Acknowledgements This work was supported by National Natural Science Foundation of China (30472098), Special Funds for 333 Projects (BRA2010138) and Industry–University–Research Institution Cooperation (BY2009141, CY2010023, CZ2009009) in Jiangsu Province and Zhenjiang City. The authors are grateful to Caleb Kesse Firempong, Jiangsu University for correcting the English. The authors also thank the Jiangsu University Ethics Committee for their kind guidance with the animal experiments. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 2–10, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2012.02.011. References [1] Pepping J. Milk thistle: Silybum marianum. Am J Health Syst Pharm 1999;56:1195–7. [2] Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol 1998;93:139–43. [3] Luper S. A review of plants used in the treatment of liver disease: part 1. Altern Med Rev 1998;3:410–21. [4] Ding T, Tian S, Zhang Z, Gu D, Chen Y, Shi Y, et al. Determination of active component in silymarin by RP-LC and LC/MS. J Pharm Biomed Anal 2001;26:155–61. [5] Kvasnicka F, Biba B, Sevcik R, Voldrich M, Kratka J. Analysis of the active components of silymarin. J Chromatogr A 2003;990:239–45. [6] Blumenthal M, Goldberg A, Brinckmann J. Herbal medicine. Expanded commission E monographs: integrative medicine communications; 2000. [7] Wachter W, Zaeske H. Process for the manufacture of flavanolignan preparation with improved release and absorbability, compositions obtainable thereby and their use for the preparation of pharmaceuticals. US Patent 6020, 384; 2000. [8] Voinovich D, Perissutti B, Magarotto L, Ceschia D, Guiotto P, Bilia A. Solid state mechanochemical simultaneous activation of the constituents of the Silybum marianum phytocomplex with crosslinked polymers. J Pharm Sci 2009;98:215–28. [9] Yan-yu X, Yun-mei S, Zhi-peng C, Qi-neng P. Preparation of silymarin proliposome: a new way to increase oral bioavailability of silymarin in beagle dogs. Int J Pharm 2006;319:162–8. [10] Wu JW, Lin LC, Hung SC, Chi CW, Tsai TH. Analysis of silibinin in rat plasma and bile for hepatobiliary excretion and oral bioavailability application. J Pharm Biomed Anal 2007;45:635–41. [11] Barzaghi N, Crema F, Gatti G, Pifferi G, Perucca E. Pharmacokinetic studies on IdB 1016, a silybin-phosphatidylcholine complex, in healthy human subjects. Eur J Drug Metab Pharmacokinet 1990;15:333–8. [12] Morazzoni P, Magistretti M, Giachetti C, Zanolo G. Comparative bioavailability of silipide, a new flavanolignan complex, in rats. Eur J Drug Metab Pharmacokinet 1992;17:39–44. [13] Sun N, Wei X, Wu B, Chen J, Lu Y, Wu W. Enhanced dissolution of silymarin/ polyvinylpyrrolidone solid dispersion pellets prepared by a one-step fluid-bed coating technique. Powder Technol 2008;182:72–80.

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