http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(6): 573–578 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.898705
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Galactosylated bovine serum albumin nanoparticles for parenteral delivery of oridonin: tissue distribution and pharmacokinetic studies Caiyun Li1, Dianrui Zhang1, Yuanyuan Guo1, Hejian Guo1, Tingting Li1, Leilei Hao1, Dandan Zheng1, Guangpu Liu1, and Qiang Zhang2 1
Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, PR China and 2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China Abstract
Keywords
Bovine serum albumin (BSA) nanoparticle is a promising drug carrier system. Oridonin (ORI)-loaded galactosylated BSA nanoparticle (ORI-GB-NP) was prepared for liver targeting delivery of ORI. This work was designed to investigate the in vitro release, in vivo pharmacokinetics and tissue distribution of ORI-GB-NP. ORI-GB-NP was prepared by the desolvation method. The particle size of ORI-GB-NP was 172.0 ± 8.3 nm with narrow size distribution. The in vitro release of ORI-GB-NP exhibited biphasic drug release pattern with an initial burst release and consequently sustained release. Pharmacokinetic analysis displayed that ORI-GB-NP and ORI-loaded BSA nanoparticle (ORI-BSA-NP) could enhance the drug plasma level and prolong the circulation time in contrast with ORI solution. Meanwhile, compared with ORI-BSA-NP, ORI-GB-NP could deliver more ORI to liver and simultaneously reduce the toxicity of ORI to heart, lung and kidney. In conclusion, ORI-GB-NP could be a promising drug delivery system for liver cancer therapy.
Bovine serum albumin nanoparticles, galactosylated, oridonin, pharmacokinetics, tissue distribution
Introduction Hepatocellular carcinoma (HCC), one of the major health problems (Rimassa and Santoro, 2010), remains the fifth most common malignancy in men and the eighth in women worldwide (Bosch et al., 2004). Chemotherapy, the main treatment of HCC so far, has been limited for its poor specificity of chemotherapeutic agents (Duan et al., 2011). Oridonin (ORI), an active diterpenoid compound extracted from the Chinese traditional medicine Rabdosia rubescens, can effectively inhibited the proliferation of many kinds of human cancer cells and it has been successfully used for the treatment against liver cancer and esophageal carcinoma in the clinical for decades (Zhang and Ren, 2003; Fujita et al., 2007). However, its poor solubility and low bioavailability limit its application in clinic (Zhang et al., 2006). In order to overcome the above pharmaceutical problems and improve the targeting behaviour of ORI to liver, it is necessary to develop an alternative formulation. Albumin-based nanoparticle (Hawkins et al., 2008; Kratz, 2008; Elzoghby et al., 2012), a biocompatible and biodegradable carrier for drugs, is expected to be well tolerated without any serious side-effects (Ibrahim et al., 2002). Nab-paclitaxel, a solvent-free formulation, can be administered without hypersensitivity premedication. Its heightened efficacy, especially on metastatic breast cancer (Lee, 2004), has been approved by Food and Drug Administration (FDA) (Coleman et al., 2011).
Address for correspondence: Dianrui Zhang, Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong Province, China. Tel: +86 531 88382015. Fax: +86 531 88382015. E-mail: [email protected]
History Received 21 January 2013 Revised 11 February 2014 Accepted 17 February 2014 Published online 2 April 2014
Galactosylated bovine serum albumin (GB) has a great potential as an effective liver targeting carrier of drugs for liver diseases (Han et al., 1999) because it can recognise and bind to the asialoglycoprotein receptor (ASGPR) which is over-expressed on the cell surface of hepatocytes (Di Stefano et al., 2004; Kim et al., 2005). In this study, galactosylated BSA nanoparticle (GB-NP) was used as a livert-argeted drug carrier for encapsulation of ORI. We expected to find whether galactosylated BSA nanoparticles could help deliver much more ORI to liver and evaluate the behaviour of ORI-GB-NP in vitro and in vivo. These results will show more information about the in vivo fate of ORIGB-NP and ORI-BSA-NP. Moreover, these preliminary data can provide references for the development of ORI formulations.
Materials and methods Materials and animals ORI (98%) was obtained from Nanjing Zelang Pharmaceutical Co. Ltd. (Nanjing, China). BSA (purity 96–99%) was purchased from BeiJing DingGuo ChangSheng Biotech. Co. Ltd. (Beijing, China). Galactosylated BSA was prepared in our lab. Glutaraldehyde was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The methanol (Tianjin Siyou Co. Ltd., Tianjin, China) was of high performance liquid chromatography (HPLC) grade. Ethanol and other reagents and solvents were of analytical reagent grade. Nitrogen was supplied by Gas Company of Jinan (Jinan, China). Water used in this study was deionised. Wistar rats (body weight 200 ± 20 g) and Kunming strain mice (body weight 20 ± 2 g), which were used for pharmacokinetic and biodistribution studies, were obtained from the Experimental Animal Center of Shandong University (Jinan, China). The
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animals were housed in cages for at least 1–2 weeks before experimentation and simultaneously fed with food and water ad libitum. Prior to the experiments all the animals were kept under fasting but with free access to water overnight. The animal care and all the experimental procedures abided by the ethics and regulations of animal experiments of Pharmaceutical Sciences, Shandong University, China.
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Preparation of ORI-loaded bovine serum albumin nanoparticle (ORI-BSA-NP) and ORI-loaded galactosylated BSA nanoparticle (ORI-GB-NP) ORI-BSA-NP and ORI-GB-NP were prepared by the desolvation technique according to our previous report (Li et al., 2013). In brief, 75 mg BSA or GB was dissolved in 5.0 mL purified water, and the pH was adjusted to 9 with NaOH. 5 mg ORI was dissolved in 1.0 mL ethanol and then the ORI solution was added into BSA or GB solution. The nanoparticles were transformed by continuous addition of the desolvating agent ethanol under stirring (600 rpm) at room temperature. The technique was modified at a rate of ethanol addition of 0.7 mL/min. Following the desolvation process, nanoparticles were stabilised by the addition of 0.5% glutaraldehyde solution. The cross-linking process was performed under continuous stirring of 24 h. The non-desolvated BSA or GB, free drug, and excipients such as ethanol and glutaraldehyde were eliminated by washing the nanoparticle solution using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cut-off of 100 kDa. Lyophilisation In order to maintain a long-term stability of the nanoparticle, lyophilisation process was necessary. The nanoparticle dispersions were filled into each Cillin-glass bottle. Then, the Cillinglass bottles were pre-frozen at 80 C. Ultra-low Temperature Freezer (DW-86L, Haier, Qingdao, China) for 12 h. The frozen nanoparticle was freeze dried for 48 h with freeze drier (FD-1000, EYELA, Tokyo, Japan) using 5% mannitol as the cryoprotectant. The freeze-dried powders were collected for further experiments. Particle size and zeta potential analysis The average particle size and zeta potential of the nanoparticle were evaluated using the DelsaÔ Nano C Particle Analyzer (Beckman Coulter, Inc., Brea, CA). Each sample was determined in triplicate. Morphology observation by transmission electron microscope (TEM) The morphology of the nanoparticle was observed by TEM (H-7000, Hitachi, Tokyo, Japan). One droplet of ORI-GB-NP was placed on the 200-mesh copper grid and then negatively stained with 2% phosphotungstic acid for 30s. The grid was dried under luminous heat bulb and examined with the TEM. In vitro drug release assay In order to create pseudo-sink conditions, the in vitro drug release of ORI from nanoparticles was determined in phosphate buffer saline (PBS, pH 7.4 containing 10% ethanol). The nanoparticles (containing 1 mg ORI) were filled into a dialysis tube (molecular cut off ¼ 12 000). The dialysis tube was immersed fully in 40 mL of the release medium and kept in an incubator at 37 ± 0.5 C with stirring at 100 rpm. At predetermined time intervals, 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh release medium to maintain constant volume
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(40 mL). The collected samples were filtered with 0.22 mm filter membrane prior to HPLC analysis. The results of triplicate measurements were used to calculate cumulative drug release. HPLC analysis HPLC analysis of ORI was performed by using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA). Twenty microliters of sample solution was injected at least three times into a Hypersil-ODS2 column (4.60 mm 250 mm, 5 mm) (Elite, Dalian, China). The mobile phase was a mixture of water and methanol in the volume ratio of 48:52. The elution rate was 1.0 mL/min and the ORI detection wavelength was set at 238 nm. Pharmacokinetics studies Pharmacokinetics of ORI-GB-NP was compared with ORI-BSANP and ORI solution (ORI-Sol). ORI solution was prepared by dissolving ORI in ethanol/ physiological saline mixture solvent (1/ 4, v/v). Experimental Wistar rats were randomly divided into three groups and four rats for each group. After administration of ORI solution, ORI-BSA-NP and ORI-GB-NP via the tail vein at a single dose of 14 mg/kg, about 0.6 mL of blood sample was collected via the sinus jugularis at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). After centrifugation at 4000 rpm for 15 min, the obtained plasma was stored at 20 C until analysis. Biodistribution studies Sixty-three Kunming strain mice were randomly divided into three groups. ORI solution, ORI-BSA-NP and ORI-GB-NP were intravenously administrated via tail vein at a dose of 20 mg kg1. Tail vein was dilation and sterilisation with ethanol solution (75%, v/v). Blood (about 0.6 mL) was collected from postorbital vein at the following time points: 0.25, 0.5, 2, 5, 8, 12, 24 h. The whole blood was immediately centrifuged at 4000 rpm for 15 min in order to get the plasma. Tissues of interest (heart, liver, spleen, lung, and kidney) were collected after the mice were sacrificed. Each organ was lightly rinsed with normal saline and dried with tissue paper. Both plasma and tissue samples were frozen at 20 C until analysis. Plasma and tissue sample analysis Liquid–liquid extraction method was used to dispose plasma samples. 500 mL (200 mL for mice) of plasma was placed in a 10 mL stoppered tube containing 0.4 mL of acetonitrile and the mixture was vortex-mixed for 1 min. Then the sample was mixed with 5 mL anhydrous ether and acetic ether (3:2, v/v) followed by vortex-mixing for 5 min and ultrasound for 10 min. Differently, for heart sample, the extracting agent was 4 mL anhydrous ether and for liver sample it was 1.5 mL acetonitrile and 3 mL acetic ether. The resulting mixture was centrifuged at 4000 rpm for 15 min. The supernatant layer was transferred into another clean tube. The precipitation layer was re-extracted with 5 mL anhydrous ether and acetic ether (3:2, v/v). After centrifuging, the supernatant layer was merged with the previous one and then evaporated under a light stream of nitrogen at 40 C. Afterwards, the residue was redissolved in 200 mL methanol and then filtered through a 0.22 mm filter. An aliquot (20 mL) of the filtrate was injected for HPLC analysis. The isolated tissue sample was weighed accurately and homogenised with 1 mL physiologic saline using a high speed shearing machine (FJ-200, Jincheng Guosheng Experimental Instrument Factory in Jiangsu, Jintan, China). Subsequently, the tissue homogenates were processed similarly as plasma samples and analysed by HPLC.
DOI: 10.3109/02652048.2014.898705
Statistics Statistical differences were assessed via student’s t-test with p50.05 as the minimal level of significance. Pharmacokinetic parameters were obtained using drug and statistics (DAS) version 2.0 software (supplied by Chinese Pharmacological Society).
Results and discussion
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Characterisation of nanoparticles In this study, the freshly prepared nanoparticles were pale yellow and homogeneous. After lyophilisation with mannitol, the powders were white and appeared flat and smooth. Figure 1 presented the TEM images of ORI-BSA-NP (Figure 1A) and ORI-GB-NP (Figure 1B). Both types of the nanoparticles were in spherical shape. The mean particle diameter of ORI-BSA-NP was 164.5 ± 6.5 nm, and its polydispersity index (PI) was 0.078 ± 0.02, while for ORI-GB-NP, the mean particle size was 172.0 ± 8.3 nm with PI of 0.074 ± 0.03. These results showed that the nanoparticles were with a unimodal and relatively narrow particle size distribution. The zeta potentials of the ORIBSA-NP and ORI-GB-NP were 25.39 ± 1.62 mV (n ¼ 3) and 31.48 ± 2.15 mV (n ¼ 3), respectively. As zeta potential values at around 30 mV signify long-term stability of aqueous dispersion (Jacobs et al., 2000), the results ensured good physical stability of the nanoparticles. In vitro release of drug In order to simulate the pH of blood, pH 7.4 phosphate buffer solution was selected as the release medium. In vitro cumulative release profiles of ORI-BSA-NP and ORI-GB-NP were compared in Figure 2. In addition, ORI released from stock solution was investigated as control. It could be found that about 80% of the drug was released after approximately 6 h. However, less than 40% of ORI was detected from the nanoparticles after 12 h. Obviously, in contrast to the drug release from stock solution, both ORI-BSA-NP and ORI-GB-NP were fitted to a biphasic drug release pattern characterised with relative burst drug release and pronounced time prolongation of drug release. At first, drug around the surface of the nanoparticles released as a relatively strong pulse with enough amounts of drug and then the nanoparticles released the drug in a sustained manner so that the drug could fight continually against cancer cell, resulting in decreased cancer cell viability (Zhao et al., 2010).
Figure 1 TEM images of the nanoparticles: ORI-BSA-NP (A) and ORI-GB-NP (B).
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Meanwhile, the drug release behaviours of ORI-BSA-NP and ORI-GB-NP were compared and they seemed almost the same. These results indicated that ORI-GB-NP might sustain controlled drug release with the erosion and degradation of the nanoparticles. Pharmacokinetic study In this study, three groups of Wistar rats were treated with ORI-Sol, ORI-BSA-NP and ORI-GB-NP at a dose of 14 mg/kg, respectively. Figure 3 displayed the ORI plasma concentrationtime curves after intravenous administration of different formulations. Compared with ORI-BSA-NP and ORI-GB-NP groups, ORI-Sol was quickly removed from the circulating system. No drug was detected after 12 h, while ORI was still present in the plasma until 24 h post-injection of ORI-BSA-NP and ORIGB-NP. Although the initial drug concentration of ORI-BSA-NP and ORI-GB-NP was lower than ORI solution, the nanoparticles showed a markedly delayed blood clearance with higher ORI concentration at later time points. Mean pharmacokinetic parameters of ORI solution, ORI-BSA-NP and ORI-GB-NP were listed in Table 1. The pharmacokinetic parameters were analysed by Student’s t-test. As could be seen from Table 1, the mean residence time (MRT) for plasma with ORI solution was found lower than ORI-GB-NP and ORI-BSA-NP, indicating that ORIGB-NP and ORI-BSA-NP increased the systemic circulation time. Moreover, the area under plasma concentration-time curve (AUC) value of ORI-GB-NP was 2.1-fold higher compared with the ORI solution and was about 1.3-fold higher compared with ORIBSA-NP, indicating a higher amount of ORI available for tissue
Figure 2 Release profiles of ORI from ORI solution, ORI-BSA-NP and ORI-GB-NP in PBS. Samples performed in triplicate; data shown were mean ± standard deviation.
Figure 3 Mean ORI concentration-time profiles of ORI in rats following i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP at a dose of 14 mg/kg (data were given as mean ± standard deviation, n ¼ 4).
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uptake (Zheng et al., 2011). In the animals injected with ORI-GB-NP and ORI-BSA-NP, plasma concentrations declined with CL of 0.224 ± 0.028 L/h/kg and 0.286 ± 0.047 L/h/kg, respectively, while the corresponding value of ORI solution was 0.678 ± 0.091 L/h/kg. The pharmacokinetic behaviour of ORI-GB-NP displayed significantly higher AUC (p50.05) and slower clearance (p50.05) than that of ORI solution. These facts given above were related to the metabolic pattern of ORI in vivo, which was in agreement with the in vitro sustained release of ORI from the nanoparticles. When ORI-Sol was injected into the vein, ORI was directly exposure to blood, resulting in faster elimination. However, the nanoparticles can serve as reservoirs for ORI in blood, which prevent the interactions between ORI and the blood components. ORI was slowly released into blood when ORI-GB-NP was gradually degraded, thus leading to prolonged residence of ORI in blood circulation, slower clearance and higher AUC. It could be concluded that ORI-GB-NP could improve the availability of ORI by prolonging drug retention and more tissue uptake in vivo. Table 1. Mean pharmacokinetic parameters of ORI after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP (mean ± SD, n ¼ 4). Parameter
Unit
ORI solution
ORI-BSA-NP
ORI-GB-NP
AUC(01) MRT(01) CL
mg/Lh h L/h/kg
29.508 ± 2.361 2.558 ± 0.152 0.678 ± 0.091
48.958 ± 3.414 5.649 ± 0.201 0.286 ± 0.047
62.423 ± 2.572 6.574 ± 0.244 0.224 ± 0.028
Biodistribution study The concentration of ORI in plasma and tissues were investigated after intravenous administration of ORI solution, ORI-BSA-NP and ORI-GB-NP in mice. The concentration-time courses of ORI were shown in Figure 4(A–F). As shown in Figure 4(A), concentration of ORI in plasma after injection with ORI-BSANP and ORI-GB-NP were both higher than that of ORI-Sol at the later time points, which was in agreement with the result of pharmacokinetics in rats. The kinetic curves of ORI-BSA-NP and ORI-GB-NP in tissues were markedly different from that of ORISol. The levels of ORI concentration of ORI-BSA-NP and ORIGB-NP were lower than that of ORI-Sol in all tissues except the liver and spleen. Especially, the level of ORI concentration of ORI-GB-NP was markedly higher than that of ORI-BSA-NP and ORI-Sol in liver. The AUC, MRT and targeting parameter value (rce ) of tested organs were given in Table 2. The parameter rce, (defined rce1 as AUC(01)(ORI-BSA-NP/ORI-Sol) and rce2 as AUC(01)(ORI-GB-NP/ORI-Sol)) which could depict the relative targeting efficacy of two delivery systems on one tissue, was introduced to evaluate the targeting efficiency of ORI-BSA-NP and ORI-GB-NP. As shown in Table 2, the rce1 of ORI-BSA-NP in listed tissues in a descending order was liver (3.48 ± 0.86)4 spleen (2.88 ± 0.57)4plasma (2.01 ± 0.24)4heart (0.94 ± 0.02) 4kidney (0.89 ± 0.01)4lung (0.79 ± 0.01), while the rce2 of ORIGB-NP in listed tissues in a descending order was liver (5.85 ± 1.24)4plasma (2.47 ± 0.83)4spleen (1.98 ± 0.78)4heart (0.87 ± 0.03)4kidney (0.68 ± 0.03) ¼lung (0.68 ± 0.04). The rce of ORI-BSA-NP and ORI-GB-NP for liver and spleen were all
Figure 4 Mean ORI concentration-time profiles in organs after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP to mice at a dose of 20 mg/kg (data were given as mean ± standard deviation, n ¼ 3).
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Table 2. AUC, MRT , rCe and TeQ value in plasma and different tissues of mice after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP (mean ± SD, n¼3).
Group
Organ
MRT(01) (h)
AUC(01) (mg/Lh)
rCe
* TQ e (%)
ORI solution
Plasma Heart Liver Spleen Lung Kidney
2.59 ± 0.63 3.84 ± 0.52 4.87 ± 1.11 6.45 ± 1.58 3.82 ± 0.73 4.14 ± 0.65
36.66 ± 2.89 91.00 ± 4.14 77.62 ± 2.72 59.34 ± 1.49 43.54 ± 1.32 76.18 ± 3.29
– – – – – –
35.25 ± 1.13 5.06 ± 0.18 42.14 ± 2.74 3.01 ± 0.08 3.64 ± 0.11 10.89 ± 0.92
ORIPlasma 6.37 ± 1.16 73.74 ± 3.78 2.01 ± 0.24 28.36 ± 2.12 BSA-NP Heart 5.70 ± 1.23 85.14 ± 4.20 0.94 ± 0.02 1.87 ± 0.23 Liver 11.83 ± 2.04 269.99 ± 6.28 3.48 ± 0.86 61.52 ± 3.58 Spleen 7.64 ± 1.84 171.00 ± 5.53 2.88 ± 0.57 3.15 ± 0.41 Lung 5.17 ± 1.00 34.19 ± 1.35 0.79 ± 0.01 1.14 ± 0.25 Kidney 5.88 ±1.27 68.17 ± 2.03 0.89 ± 0.01 3.96 ± 0.77 ORIGB-NP
Plasma 7.85 ± 1.09 90.61 ± 5.23 2.47 ± 0.83 25.88 ± 2.44 Heart 6.35 ± 1.11 78.71 ± 3.77 0.87 ± 0.03 1.24 ± 0.07 Liver 15.80 ± 3.55 453.85 ± 8.12 5.85 ± 1.24 68.53 ± 4.64 Spleen 6.95 ± 2.03 117.30 ± 7.36 1.98 ± 0.78 1.56 ± 0.15 Lung 5.87 ± 1.89 29.74 ± 2.46 0.68 ± 0.04 0.77 ± 0.05 Kidney 5.95 ± 1.45 51.51 ± 3.39 0.68 ± 0.03 2.03 ± 0.34
Note: *TQ e ¼ (AUQ01)i/(AUQ01)i ; AUQ01¼ AUC01Wi.
greater than 1, indicating that the in vivo behaviours of ORI-BSANP and ORI-GB-NP were significantly changed and the two kinds of nanoparticles were both more efficient to deliver ORI to liver and spleen compared with the ORI solution. For ORI-BSA-NP, this might possibly due to the passive targeting ability of BSA nanoparticles for reticulate endothelial system (RES) organs such as liver and spleen (Yang et al., 2007). Furthermore, the rce of liver for ORI-GB-NP was 5.85 ± 1.24 while for ORI-BSA-NP was 3.48 ± 0.86, and the re of heart, lung and kidney for ORI-GB-NP were all lower than that of ORI-BSA-NP. TQ e of ORI-Sol, ORI-BSA-NP and ORI-GB-NP calculated by AUQ (quality of drugs within the tissues – the area under the curve) were listed in Table 2. For ORI-Sol, 42.14%, 35.25%, 10.89% and 5.06% were distributed in liver, plasma, kidney and heart, respectively. Whereas, for ORI-GB-NP, 68.53%, 25.88%, 2.03% and 1.56% were distributed in liver, plasma, kidney and spleen, respectively. Conspicuously, ORI-GB-NP enhanced the accumulation of ORI in liver and reduced the accumulation in heart and kidney, which was possibly attributed to the liver-targeted delivery route of ORIGB-NP. Figure 5(A–C) presented relative distribution percentage of ORI in plasma, heart, liver, spleen, lung and kidney at different time following i.v. administration of ORI-Sol, ORI-BSA-NP and ORI-GB-NP. For injection of ORI-GB-NP at 12 h, 65.03%, 7.62% and 5.82% of ORI were distributed in liver, heart and kidney, respectively. For the same time period of ORI-BSA-NP, 58.39%, 8.33% and 8.38% of ORI were distributed in the above organs, respectively. Whereas, for ORI-Sol, 21.51%, 18.62% and 19.29% of ORI were distributed in the above organs, respectively. These results obviously demonstrated that ORI-GB-NP was superior to ORI-Sol as well as ORI-BSA-NP since ORI-GB-NP could deliver more ORI to liver and at the same time reduce the toxicity to heart and kidney. As for the reasons, besides the size-dependent passive targeting of the nanoparticles, the active targeting effects of ORI-GB-NP played an important role for the phenomenon (Zheng et al., 2012). The active targeting effect was mediated by the ASGPR, located on the hepatocytes and hepatoma carcinoma cell membrane (Tozawa et al., 2001), which could specifically
Figure 5 Relative distribution percentage of ORI in plasma and tissues at different time following i.v. administration of ORI solution (A), ORI-BSA-NP (B) and ORI-GB-NP(C) in mice.
recognise the galactose on the nanoparticles, thus facilitating more drug delivery to liver. These data demonstrated that with the aid of ORI-GB-NP, more ORI could be accumulated in liver with certain specificity whereas a subsequent reduced distribution in other organs, which was beneficial to therapy of liver cancer.
Conclusion In the present work, ORI-GB-NP was successfully prepared as a liver-targeted drug delivery system. In summary, in vitro release profiles indicated that the release of ORI from ORI-GB-NP exhibited a sustained release behaviour. A similar phenomenon was also observed in the in vivo pharmacokinetic study. Compared with ORI-BSA-NP and ORI solution, ORI-GB-NP dislayed higher AUC value and a prolonged residence of drug in blood circulation. Moreover, higher drug concentration, longer residence time of drug and greater relative distribution percentage
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in liver demonstrated that ORI-GB-NP could directly target to liver and reduce the side effects of ORI to other organs. In conclusion, ORI-loaded galactosylated BSA nanoparticles targeting hepatocytes by both active and passive mechanisms, might hold great promise to deliver ORI for therapy of HCC.
Acknowledgements The authors are thankful to the National Basic Research Programme of China (973 Programme) for providing the financial support.
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Declaration of interest This study was supported by the National Basic Research Programme of China (2009CB930300).
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Galactosylated bovine serum albumin nanoparticles for parenteral delivery of oridonin: tissue distribution and pharmacokinetic studies Caiyun Li1, Dianrui Zhang1, Yuanyuan Guo1, Hejian Guo1, Tingting Li1, Leilei Hao1, Dandan Zheng1, Guangpu Liu1, and Qiang Zhang2 1
Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, PR China and 2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China Abstract
Keywords
Bovine serum albumin (BSA) nanoparticle is a promising drug carrier system. Oridonin (ORI)-loaded galactosylated BSA nanoparticle (ORI-GB-NP) was prepared for liver targeting delivery of ORI. This work was designed to investigate the in vitro release, in vivo pharmacokinetics and tissue distribution of ORI-GB-NP. ORI-GB-NP was prepared by the desolvation method. The particle size of ORI-GB-NP was 172.0 ± 8.3 nm with narrow size distribution. The in vitro release of ORI-GB-NP exhibited biphasic drug release pattern with an initial burst release and consequently sustained release. Pharmacokinetic analysis displayed that ORI-GB-NP and ORI-loaded BSA nanoparticle (ORI-BSA-NP) could enhance the drug plasma level and prolong the circulation time in contrast with ORI solution. Meanwhile, compared with ORI-BSA-NP, ORI-GB-NP could deliver more ORI to liver and simultaneously reduce the toxicity of ORI to heart, lung and kidney. In conclusion, ORI-GB-NP could be a promising drug delivery system for liver cancer therapy.
Bovine serum albumin nanoparticles, galactosylated, oridonin, pharmacokinetics, tissue distribution
Introduction Hepatocellular carcinoma (HCC), one of the major health problems (Rimassa and Santoro, 2010), remains the fifth most common malignancy in men and the eighth in women worldwide (Bosch et al., 2004). Chemotherapy, the main treatment of HCC so far, has been limited for its poor specificity of chemotherapeutic agents (Duan et al., 2011). Oridonin (ORI), an active diterpenoid compound extracted from the Chinese traditional medicine Rabdosia rubescens, can effectively inhibited the proliferation of many kinds of human cancer cells and it has been successfully used for the treatment against liver cancer and esophageal carcinoma in the clinical for decades (Zhang and Ren, 2003; Fujita et al., 2007). However, its poor solubility and low bioavailability limit its application in clinic (Zhang et al., 2006). In order to overcome the above pharmaceutical problems and improve the targeting behaviour of ORI to liver, it is necessary to develop an alternative formulation. Albumin-based nanoparticle (Hawkins et al., 2008; Kratz, 2008; Elzoghby et al., 2012), a biocompatible and biodegradable carrier for drugs, is expected to be well tolerated without any serious side-effects (Ibrahim et al., 2002). Nab-paclitaxel, a solvent-free formulation, can be administered without hypersensitivity premedication. Its heightened efficacy, especially on metastatic breast cancer (Lee, 2004), has been approved by Food and Drug Administration (FDA) (Coleman et al., 2011).
Address for correspondence: Dianrui Zhang, Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong Province, China. Tel: +86 531 88382015. Fax: +86 531 88382015. E-mail: [email protected]
History Received 21 January 2013 Revised 11 February 2014 Accepted 17 February 2014 Published online 2 April 2014
Galactosylated bovine serum albumin (GB) has a great potential as an effective liver targeting carrier of drugs for liver diseases (Han et al., 1999) because it can recognise and bind to the asialoglycoprotein receptor (ASGPR) which is over-expressed on the cell surface of hepatocytes (Di Stefano et al., 2004; Kim et al., 2005). In this study, galactosylated BSA nanoparticle (GB-NP) was used as a livert-argeted drug carrier for encapsulation of ORI. We expected to find whether galactosylated BSA nanoparticles could help deliver much more ORI to liver and evaluate the behaviour of ORI-GB-NP in vitro and in vivo. These results will show more information about the in vivo fate of ORIGB-NP and ORI-BSA-NP. Moreover, these preliminary data can provide references for the development of ORI formulations.
Materials and methods Materials and animals ORI (98%) was obtained from Nanjing Zelang Pharmaceutical Co. Ltd. (Nanjing, China). BSA (purity 96–99%) was purchased from BeiJing DingGuo ChangSheng Biotech. Co. Ltd. (Beijing, China). Galactosylated BSA was prepared in our lab. Glutaraldehyde was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The methanol (Tianjin Siyou Co. Ltd., Tianjin, China) was of high performance liquid chromatography (HPLC) grade. Ethanol and other reagents and solvents were of analytical reagent grade. Nitrogen was supplied by Gas Company of Jinan (Jinan, China). Water used in this study was deionised. Wistar rats (body weight 200 ± 20 g) and Kunming strain mice (body weight 20 ± 2 g), which were used for pharmacokinetic and biodistribution studies, were obtained from the Experimental Animal Center of Shandong University (Jinan, China). The
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animals were housed in cages for at least 1–2 weeks before experimentation and simultaneously fed with food and water ad libitum. Prior to the experiments all the animals were kept under fasting but with free access to water overnight. The animal care and all the experimental procedures abided by the ethics and regulations of animal experiments of Pharmaceutical Sciences, Shandong University, China.
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Preparation of ORI-loaded bovine serum albumin nanoparticle (ORI-BSA-NP) and ORI-loaded galactosylated BSA nanoparticle (ORI-GB-NP) ORI-BSA-NP and ORI-GB-NP were prepared by the desolvation technique according to our previous report (Li et al., 2013). In brief, 75 mg BSA or GB was dissolved in 5.0 mL purified water, and the pH was adjusted to 9 with NaOH. 5 mg ORI was dissolved in 1.0 mL ethanol and then the ORI solution was added into BSA or GB solution. The nanoparticles were transformed by continuous addition of the desolvating agent ethanol under stirring (600 rpm) at room temperature. The technique was modified at a rate of ethanol addition of 0.7 mL/min. Following the desolvation process, nanoparticles were stabilised by the addition of 0.5% glutaraldehyde solution. The cross-linking process was performed under continuous stirring of 24 h. The non-desolvated BSA or GB, free drug, and excipients such as ethanol and glutaraldehyde were eliminated by washing the nanoparticle solution using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cut-off of 100 kDa. Lyophilisation In order to maintain a long-term stability of the nanoparticle, lyophilisation process was necessary. The nanoparticle dispersions were filled into each Cillin-glass bottle. Then, the Cillinglass bottles were pre-frozen at 80 C. Ultra-low Temperature Freezer (DW-86L, Haier, Qingdao, China) for 12 h. The frozen nanoparticle was freeze dried for 48 h with freeze drier (FD-1000, EYELA, Tokyo, Japan) using 5% mannitol as the cryoprotectant. The freeze-dried powders were collected for further experiments. Particle size and zeta potential analysis The average particle size and zeta potential of the nanoparticle were evaluated using the DelsaÔ Nano C Particle Analyzer (Beckman Coulter, Inc., Brea, CA). Each sample was determined in triplicate. Morphology observation by transmission electron microscope (TEM) The morphology of the nanoparticle was observed by TEM (H-7000, Hitachi, Tokyo, Japan). One droplet of ORI-GB-NP was placed on the 200-mesh copper grid and then negatively stained with 2% phosphotungstic acid for 30s. The grid was dried under luminous heat bulb and examined with the TEM. In vitro drug release assay In order to create pseudo-sink conditions, the in vitro drug release of ORI from nanoparticles was determined in phosphate buffer saline (PBS, pH 7.4 containing 10% ethanol). The nanoparticles (containing 1 mg ORI) were filled into a dialysis tube (molecular cut off ¼ 12 000). The dialysis tube was immersed fully in 40 mL of the release medium and kept in an incubator at 37 ± 0.5 C with stirring at 100 rpm. At predetermined time intervals, 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh release medium to maintain constant volume
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(40 mL). The collected samples were filtered with 0.22 mm filter membrane prior to HPLC analysis. The results of triplicate measurements were used to calculate cumulative drug release. HPLC analysis HPLC analysis of ORI was performed by using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA). Twenty microliters of sample solution was injected at least three times into a Hypersil-ODS2 column (4.60 mm 250 mm, 5 mm) (Elite, Dalian, China). The mobile phase was a mixture of water and methanol in the volume ratio of 48:52. The elution rate was 1.0 mL/min and the ORI detection wavelength was set at 238 nm. Pharmacokinetics studies Pharmacokinetics of ORI-GB-NP was compared with ORI-BSANP and ORI solution (ORI-Sol). ORI solution was prepared by dissolving ORI in ethanol/ physiological saline mixture solvent (1/ 4, v/v). Experimental Wistar rats were randomly divided into three groups and four rats for each group. After administration of ORI solution, ORI-BSA-NP and ORI-GB-NP via the tail vein at a single dose of 14 mg/kg, about 0.6 mL of blood sample was collected via the sinus jugularis at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). After centrifugation at 4000 rpm for 15 min, the obtained plasma was stored at 20 C until analysis. Biodistribution studies Sixty-three Kunming strain mice were randomly divided into three groups. ORI solution, ORI-BSA-NP and ORI-GB-NP were intravenously administrated via tail vein at a dose of 20 mg kg1. Tail vein was dilation and sterilisation with ethanol solution (75%, v/v). Blood (about 0.6 mL) was collected from postorbital vein at the following time points: 0.25, 0.5, 2, 5, 8, 12, 24 h. The whole blood was immediately centrifuged at 4000 rpm for 15 min in order to get the plasma. Tissues of interest (heart, liver, spleen, lung, and kidney) were collected after the mice were sacrificed. Each organ was lightly rinsed with normal saline and dried with tissue paper. Both plasma and tissue samples were frozen at 20 C until analysis. Plasma and tissue sample analysis Liquid–liquid extraction method was used to dispose plasma samples. 500 mL (200 mL for mice) of plasma was placed in a 10 mL stoppered tube containing 0.4 mL of acetonitrile and the mixture was vortex-mixed for 1 min. Then the sample was mixed with 5 mL anhydrous ether and acetic ether (3:2, v/v) followed by vortex-mixing for 5 min and ultrasound for 10 min. Differently, for heart sample, the extracting agent was 4 mL anhydrous ether and for liver sample it was 1.5 mL acetonitrile and 3 mL acetic ether. The resulting mixture was centrifuged at 4000 rpm for 15 min. The supernatant layer was transferred into another clean tube. The precipitation layer was re-extracted with 5 mL anhydrous ether and acetic ether (3:2, v/v). After centrifuging, the supernatant layer was merged with the previous one and then evaporated under a light stream of nitrogen at 40 C. Afterwards, the residue was redissolved in 200 mL methanol and then filtered through a 0.22 mm filter. An aliquot (20 mL) of the filtrate was injected for HPLC analysis. The isolated tissue sample was weighed accurately and homogenised with 1 mL physiologic saline using a high speed shearing machine (FJ-200, Jincheng Guosheng Experimental Instrument Factory in Jiangsu, Jintan, China). Subsequently, the tissue homogenates were processed similarly as plasma samples and analysed by HPLC.
DOI: 10.3109/02652048.2014.898705
Statistics Statistical differences were assessed via student’s t-test with p50.05 as the minimal level of significance. Pharmacokinetic parameters were obtained using drug and statistics (DAS) version 2.0 software (supplied by Chinese Pharmacological Society).
Results and discussion
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Characterisation of nanoparticles In this study, the freshly prepared nanoparticles were pale yellow and homogeneous. After lyophilisation with mannitol, the powders were white and appeared flat and smooth. Figure 1 presented the TEM images of ORI-BSA-NP (Figure 1A) and ORI-GB-NP (Figure 1B). Both types of the nanoparticles were in spherical shape. The mean particle diameter of ORI-BSA-NP was 164.5 ± 6.5 nm, and its polydispersity index (PI) was 0.078 ± 0.02, while for ORI-GB-NP, the mean particle size was 172.0 ± 8.3 nm with PI of 0.074 ± 0.03. These results showed that the nanoparticles were with a unimodal and relatively narrow particle size distribution. The zeta potentials of the ORIBSA-NP and ORI-GB-NP were 25.39 ± 1.62 mV (n ¼ 3) and 31.48 ± 2.15 mV (n ¼ 3), respectively. As zeta potential values at around 30 mV signify long-term stability of aqueous dispersion (Jacobs et al., 2000), the results ensured good physical stability of the nanoparticles. In vitro release of drug In order to simulate the pH of blood, pH 7.4 phosphate buffer solution was selected as the release medium. In vitro cumulative release profiles of ORI-BSA-NP and ORI-GB-NP were compared in Figure 2. In addition, ORI released from stock solution was investigated as control. It could be found that about 80% of the drug was released after approximately 6 h. However, less than 40% of ORI was detected from the nanoparticles after 12 h. Obviously, in contrast to the drug release from stock solution, both ORI-BSA-NP and ORI-GB-NP were fitted to a biphasic drug release pattern characterised with relative burst drug release and pronounced time prolongation of drug release. At first, drug around the surface of the nanoparticles released as a relatively strong pulse with enough amounts of drug and then the nanoparticles released the drug in a sustained manner so that the drug could fight continually against cancer cell, resulting in decreased cancer cell viability (Zhao et al., 2010).
Figure 1 TEM images of the nanoparticles: ORI-BSA-NP (A) and ORI-GB-NP (B).
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Meanwhile, the drug release behaviours of ORI-BSA-NP and ORI-GB-NP were compared and they seemed almost the same. These results indicated that ORI-GB-NP might sustain controlled drug release with the erosion and degradation of the nanoparticles. Pharmacokinetic study In this study, three groups of Wistar rats were treated with ORI-Sol, ORI-BSA-NP and ORI-GB-NP at a dose of 14 mg/kg, respectively. Figure 3 displayed the ORI plasma concentrationtime curves after intravenous administration of different formulations. Compared with ORI-BSA-NP and ORI-GB-NP groups, ORI-Sol was quickly removed from the circulating system. No drug was detected after 12 h, while ORI was still present in the plasma until 24 h post-injection of ORI-BSA-NP and ORIGB-NP. Although the initial drug concentration of ORI-BSA-NP and ORI-GB-NP was lower than ORI solution, the nanoparticles showed a markedly delayed blood clearance with higher ORI concentration at later time points. Mean pharmacokinetic parameters of ORI solution, ORI-BSA-NP and ORI-GB-NP were listed in Table 1. The pharmacokinetic parameters were analysed by Student’s t-test. As could be seen from Table 1, the mean residence time (MRT) for plasma with ORI solution was found lower than ORI-GB-NP and ORI-BSA-NP, indicating that ORIGB-NP and ORI-BSA-NP increased the systemic circulation time. Moreover, the area under plasma concentration-time curve (AUC) value of ORI-GB-NP was 2.1-fold higher compared with the ORI solution and was about 1.3-fold higher compared with ORIBSA-NP, indicating a higher amount of ORI available for tissue
Figure 2 Release profiles of ORI from ORI solution, ORI-BSA-NP and ORI-GB-NP in PBS. Samples performed in triplicate; data shown were mean ± standard deviation.
Figure 3 Mean ORI concentration-time profiles of ORI in rats following i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP at a dose of 14 mg/kg (data were given as mean ± standard deviation, n ¼ 4).
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uptake (Zheng et al., 2011). In the animals injected with ORI-GB-NP and ORI-BSA-NP, plasma concentrations declined with CL of 0.224 ± 0.028 L/h/kg and 0.286 ± 0.047 L/h/kg, respectively, while the corresponding value of ORI solution was 0.678 ± 0.091 L/h/kg. The pharmacokinetic behaviour of ORI-GB-NP displayed significantly higher AUC (p50.05) and slower clearance (p50.05) than that of ORI solution. These facts given above were related to the metabolic pattern of ORI in vivo, which was in agreement with the in vitro sustained release of ORI from the nanoparticles. When ORI-Sol was injected into the vein, ORI was directly exposure to blood, resulting in faster elimination. However, the nanoparticles can serve as reservoirs for ORI in blood, which prevent the interactions between ORI and the blood components. ORI was slowly released into blood when ORI-GB-NP was gradually degraded, thus leading to prolonged residence of ORI in blood circulation, slower clearance and higher AUC. It could be concluded that ORI-GB-NP could improve the availability of ORI by prolonging drug retention and more tissue uptake in vivo. Table 1. Mean pharmacokinetic parameters of ORI after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP (mean ± SD, n ¼ 4). Parameter
Unit
ORI solution
ORI-BSA-NP
ORI-GB-NP
AUC(01) MRT(01) CL
mg/Lh h L/h/kg
29.508 ± 2.361 2.558 ± 0.152 0.678 ± 0.091
48.958 ± 3.414 5.649 ± 0.201 0.286 ± 0.047
62.423 ± 2.572 6.574 ± 0.244 0.224 ± 0.028
Biodistribution study The concentration of ORI in plasma and tissues were investigated after intravenous administration of ORI solution, ORI-BSA-NP and ORI-GB-NP in mice. The concentration-time courses of ORI were shown in Figure 4(A–F). As shown in Figure 4(A), concentration of ORI in plasma after injection with ORI-BSANP and ORI-GB-NP were both higher than that of ORI-Sol at the later time points, which was in agreement with the result of pharmacokinetics in rats. The kinetic curves of ORI-BSA-NP and ORI-GB-NP in tissues were markedly different from that of ORISol. The levels of ORI concentration of ORI-BSA-NP and ORIGB-NP were lower than that of ORI-Sol in all tissues except the liver and spleen. Especially, the level of ORI concentration of ORI-GB-NP was markedly higher than that of ORI-BSA-NP and ORI-Sol in liver. The AUC, MRT and targeting parameter value (rce ) of tested organs were given in Table 2. The parameter rce, (defined rce1 as AUC(01)(ORI-BSA-NP/ORI-Sol) and rce2 as AUC(01)(ORI-GB-NP/ORI-Sol)) which could depict the relative targeting efficacy of two delivery systems on one tissue, was introduced to evaluate the targeting efficiency of ORI-BSA-NP and ORI-GB-NP. As shown in Table 2, the rce1 of ORI-BSA-NP in listed tissues in a descending order was liver (3.48 ± 0.86)4 spleen (2.88 ± 0.57)4plasma (2.01 ± 0.24)4heart (0.94 ± 0.02) 4kidney (0.89 ± 0.01)4lung (0.79 ± 0.01), while the rce2 of ORIGB-NP in listed tissues in a descending order was liver (5.85 ± 1.24)4plasma (2.47 ± 0.83)4spleen (1.98 ± 0.78)4heart (0.87 ± 0.03)4kidney (0.68 ± 0.03) ¼lung (0.68 ± 0.04). The rce of ORI-BSA-NP and ORI-GB-NP for liver and spleen were all
Figure 4 Mean ORI concentration-time profiles in organs after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP to mice at a dose of 20 mg/kg (data were given as mean ± standard deviation, n ¼ 3).
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Table 2. AUC, MRT , rCe and TeQ value in plasma and different tissues of mice after i.v. administration of ORI solution, ORI-BSA-NP and ORI-GB-NP (mean ± SD, n¼3).
Group
Organ
MRT(01) (h)
AUC(01) (mg/Lh)
rCe
* TQ e (%)
ORI solution
Plasma Heart Liver Spleen Lung Kidney
2.59 ± 0.63 3.84 ± 0.52 4.87 ± 1.11 6.45 ± 1.58 3.82 ± 0.73 4.14 ± 0.65
36.66 ± 2.89 91.00 ± 4.14 77.62 ± 2.72 59.34 ± 1.49 43.54 ± 1.32 76.18 ± 3.29
– – – – – –
35.25 ± 1.13 5.06 ± 0.18 42.14 ± 2.74 3.01 ± 0.08 3.64 ± 0.11 10.89 ± 0.92
ORIPlasma 6.37 ± 1.16 73.74 ± 3.78 2.01 ± 0.24 28.36 ± 2.12 BSA-NP Heart 5.70 ± 1.23 85.14 ± 4.20 0.94 ± 0.02 1.87 ± 0.23 Liver 11.83 ± 2.04 269.99 ± 6.28 3.48 ± 0.86 61.52 ± 3.58 Spleen 7.64 ± 1.84 171.00 ± 5.53 2.88 ± 0.57 3.15 ± 0.41 Lung 5.17 ± 1.00 34.19 ± 1.35 0.79 ± 0.01 1.14 ± 0.25 Kidney 5.88 ±1.27 68.17 ± 2.03 0.89 ± 0.01 3.96 ± 0.77 ORIGB-NP
Plasma 7.85 ± 1.09 90.61 ± 5.23 2.47 ± 0.83 25.88 ± 2.44 Heart 6.35 ± 1.11 78.71 ± 3.77 0.87 ± 0.03 1.24 ± 0.07 Liver 15.80 ± 3.55 453.85 ± 8.12 5.85 ± 1.24 68.53 ± 4.64 Spleen 6.95 ± 2.03 117.30 ± 7.36 1.98 ± 0.78 1.56 ± 0.15 Lung 5.87 ± 1.89 29.74 ± 2.46 0.68 ± 0.04 0.77 ± 0.05 Kidney 5.95 ± 1.45 51.51 ± 3.39 0.68 ± 0.03 2.03 ± 0.34
Note: *TQ e ¼ (AUQ01)i/(AUQ01)i ; AUQ01¼ AUC01Wi.
greater than 1, indicating that the in vivo behaviours of ORI-BSANP and ORI-GB-NP were significantly changed and the two kinds of nanoparticles were both more efficient to deliver ORI to liver and spleen compared with the ORI solution. For ORI-BSA-NP, this might possibly due to the passive targeting ability of BSA nanoparticles for reticulate endothelial system (RES) organs such as liver and spleen (Yang et al., 2007). Furthermore, the rce of liver for ORI-GB-NP was 5.85 ± 1.24 while for ORI-BSA-NP was 3.48 ± 0.86, and the re of heart, lung and kidney for ORI-GB-NP were all lower than that of ORI-BSA-NP. TQ e of ORI-Sol, ORI-BSA-NP and ORI-GB-NP calculated by AUQ (quality of drugs within the tissues – the area under the curve) were listed in Table 2. For ORI-Sol, 42.14%, 35.25%, 10.89% and 5.06% were distributed in liver, plasma, kidney and heart, respectively. Whereas, for ORI-GB-NP, 68.53%, 25.88%, 2.03% and 1.56% were distributed in liver, plasma, kidney and spleen, respectively. Conspicuously, ORI-GB-NP enhanced the accumulation of ORI in liver and reduced the accumulation in heart and kidney, which was possibly attributed to the liver-targeted delivery route of ORIGB-NP. Figure 5(A–C) presented relative distribution percentage of ORI in plasma, heart, liver, spleen, lung and kidney at different time following i.v. administration of ORI-Sol, ORI-BSA-NP and ORI-GB-NP. For injection of ORI-GB-NP at 12 h, 65.03%, 7.62% and 5.82% of ORI were distributed in liver, heart and kidney, respectively. For the same time period of ORI-BSA-NP, 58.39%, 8.33% and 8.38% of ORI were distributed in the above organs, respectively. Whereas, for ORI-Sol, 21.51%, 18.62% and 19.29% of ORI were distributed in the above organs, respectively. These results obviously demonstrated that ORI-GB-NP was superior to ORI-Sol as well as ORI-BSA-NP since ORI-GB-NP could deliver more ORI to liver and at the same time reduce the toxicity to heart and kidney. As for the reasons, besides the size-dependent passive targeting of the nanoparticles, the active targeting effects of ORI-GB-NP played an important role for the phenomenon (Zheng et al., 2012). The active targeting effect was mediated by the ASGPR, located on the hepatocytes and hepatoma carcinoma cell membrane (Tozawa et al., 2001), which could specifically
Figure 5 Relative distribution percentage of ORI in plasma and tissues at different time following i.v. administration of ORI solution (A), ORI-BSA-NP (B) and ORI-GB-NP(C) in mice.
recognise the galactose on the nanoparticles, thus facilitating more drug delivery to liver. These data demonstrated that with the aid of ORI-GB-NP, more ORI could be accumulated in liver with certain specificity whereas a subsequent reduced distribution in other organs, which was beneficial to therapy of liver cancer.
Conclusion In the present work, ORI-GB-NP was successfully prepared as a liver-targeted drug delivery system. In summary, in vitro release profiles indicated that the release of ORI from ORI-GB-NP exhibited a sustained release behaviour. A similar phenomenon was also observed in the in vivo pharmacokinetic study. Compared with ORI-BSA-NP and ORI solution, ORI-GB-NP dislayed higher AUC value and a prolonged residence of drug in blood circulation. Moreover, higher drug concentration, longer residence time of drug and greater relative distribution percentage
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in liver demonstrated that ORI-GB-NP could directly target to liver and reduce the side effects of ORI to other organs. In conclusion, ORI-loaded galactosylated BSA nanoparticles targeting hepatocytes by both active and passive mechanisms, might hold great promise to deliver ORI for therapy of HCC.
Acknowledgements The authors are thankful to the National Basic Research Programme of China (973 Programme) for providing the financial support.
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Declaration of interest This study was supported by the National Basic Research Programme of China (2009CB930300).
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