Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects

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International Journal of Pharmaceutics xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

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Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects

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Huan Yu a , Lirong Teng a , Qingfan Meng a , Yuhuan Li a , Xiaocheng Sun a , Jiahui Lu a , Robert J. Lee a,b , Lesheng Teng a,b,∗ a b

Institute of Life Sciences, Jilin University, Changchun, Jilin, China Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA

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a r t i c l e

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a b s t r a c t

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Article history: Received 17 February 2013 Received in revised form 1 April 2013 Accepted 22 April 2013 Available online xxx

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Keywords: Ginsenoside Rg3 Liposome Antitumor activity MVD Cytotoxicity

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1. Introduction

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The Ginsenoside Rg3 has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of it, our aim was to prepare Rg3 liposome (L-Rg3) and optimize the preparation conditions and to investigate further whether liposome could promote the anticancer activity of it. L-Rg3 was prepared using a film-dispersion method and the preparation conditions of it were optimized with response surface methodology (RSM). The mean experimental encapsulation efficiency (EE) 82.47% was close to the predicted results 89.69%; therefore, the optimized preparation condition is reliable. We evaluated the cytotoxicity, pharmacokinetics, biodistribution and antitumor activities of L-Rg3. HepG2 and A549 cells were treated with Rg3 solution and L-Rg3 at different concentrations in vitro. Pharmacokinetics and biodistribution were carried out in Wistar rats. Tumor model was prepared by inoculating a suspension of A549 cells into BALB/c nude mice. The mice were divided into Saline, Rg3 solution, or L-Rg3 group given by i.p. injection. Survival of the mice, tumor volume was monitored. In addition, CD34 immunohistochemical analysis was used for measuring microvessel density (MVD) of the tumor tissues. The cytotoxicity and ratio of inhibition tumor of L-Rg3 group were significantly higher than the Rg3 solution group. MVD value in the Rg3 solution and L-Rg3 group decreased, especially in the L-Rg3 group. Compared to Rg3 solution, the L-Rg3 showed increased Cmax and AUC of Rg3 by 1.19- and 1.52-fold, respectively. This liposome could potentially produce viable clinical strategies for improved anticancer activity of Rg3 for treatment of cancer. © 2013 Published by Elsevier B.V.

Ginseng is a traditional herbal medicine with a wide spectrum of pharmacological effects (Ma et al., 2008), such as tonic, immunomodulatory, anti-mutagenic, adaptogenic, and antiaging activities. Therefore, it has been used as traditional medicine in the orient for a long time (Kiefer and Pantuso, 2003; Lee et al., 2005). The triterpene glycosides known as ginsenosides play a major role in many of its medicinal effects (Attele et al., 1999; Yuan et al., 2002). Studies have shown that its active ingredient Ginsenoside Rg3 can inhibit the growth of several cancer cell lines (Kim et al., 2004; Lu et al., 2008; Luo et al., 2008; Xu et al., 2007, 2008; Zhang et al., 2008). Studies have shown Ginsenoside Rg3 as a relatively safe drug with anticancer activity both in vitro and in vivo (Huang et al., 2009).

∗ Corresponding author at: Institute of Life Sciences, Jilin University, No. 2699, Qianjin Avenue, Changchun City, Jilin Province, China. Tel.: +86 431 85168646; fax: +86 431 85168637. E-mail addresses: [email protected], [email protected] (L. Teng).

It is well established that angiogenesis plays a key role in the process of solid tumor growth and infiltration (Carmeliet and Jain, 2000; Folkman, 1992); therefore, anti-angiogenic therapy is one of the most promising approaches for the treatment of cancers (Bergers and Benjamin, 2003; Jain, 2001; Lakka and Rao, 2008). Rg3 is extracted from ginseng, which is an angiogenic inhibitor (Chen et al., 2008). Rg3 (1–103 nM) dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF) (Yue et al., 2006). Ginsenoside Rg3 can significantly inhibit the metastasis of ovarian cancer. The inhibitory effect is partially due to inhibition of tumor-induced angiogenesis and decrease of invasive ability and MMP-9 expression of SKOV-3 cells (Xu et al., 2008). Li et al. evaluated the effectiveness of Ginsenoside Rg3 alone or in combination with cyclophosphamide (CPA) on tumor growth and angiogenesis in human lung cancer. The PCNALI, MVD and VEGF expression in mice of the treated groups were significantly lowered when compared with that of the control group. Additionally, the MVD of mice in groups with treatment of Ginsenoside Rg3 alone or Ginsenoside Rg3 plus CPA were lower than that in the CPA group. Tumor growth and angiogenesis in lung cancer were profoundly

0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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inhibited by Ginsenoside Rg3 alone or in combination with CPA (Li et al., 2011). However, Rg3 has poor solubility (Liu et al., 2008) and oral bioavailability (Tang et al., 2008), which limits its clinical activity. Rg3 has low solubility in waters, which means a delivery vehicle is needed if this drug is to be administered systemically. In order to improve Rg3 biodistribution in vivo, a safer and more effective formulation of Rg3 is highly desired. Liposomes contain one or more amphiphilic bilayers and an internal aqueous space, and can be used as drug carriers. Liposomes have been used to formulate a variety of drugs. Hydrophobic chemotherapeutic agents, such as paclitaxel (Zhang et al., 2005) and docetaxel (Koshkina et al., 2001), can incorporate into the liposomal bilayer, leading to improved solubility, prolonged circulation time, altered biodistribution in vivo, and reduced side-effects. Response surface methodology (RSM) is a rapid technique used to empirically derive functional relationship between one or more than one experimental response and a set of input variables (Chiang et al., 2003; Hamsaveni et al., 2001; Zhang et al., 2007). Furthermore, it may determine the optimum level of experimental factors required for the given response. RSM is widely used to optimize process parameters. In this study, Rg3 was encapsulated with liposome using a film-dispersion method and the preparation conditions of L-Rg3 were optimized with RSM. In this article, we report a liposomal formulation of Rg3 based on egg yolk phosphatidylcholine (ePC). The preparation, characterization, cytotoxicity, and anticancer potency of L-Rg3 were studied.

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2. Materials and methods

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2.1. Materials

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Egg yolk phosphatidylcholine (ePC), and cholesterol (Chol), were purchased from Avanti Polar Lipids and were used without further purification. Rg3 was purchased from Fu Sheng Pharmaceutical Limited Company, Da Lian, China. Mouse monoclonal antibody for CD34 and LSAB kit were purchased from Dako (Japan). All other reagents were analytical grade.

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2.2. Animals and tumor cell line

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Twenty-five female BALB/c nude mice, 4 weeks, 16 ± 3 g; fiftyfour male Wistar rats, 6 weeks, 200 ± 20 g were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). The experimental animals were allowed free access to water and mouse chow, and were housed under controlled environmental conditions (constant temperature, humidity, and 12 h dark/light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of the Jilin University. The human pulmonary carcinoma cell line A549 and hepatic carcinoma cell line Hepg2 were purchased from China Center for Type Culture Collection (Wuhan, China) and cultured using Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, pyruvate, glutamine and supplemented with 10% fetal bovine serum (FBS) purchased from Sigma–Aldrich (St. Louis, MO, USA) in a 5% CO2 air incubator at 37 ◦ C.

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2.3. Preparation of L-Rg3

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L-Rg3 was prepared by polycarbonate membrane extrusion method, as described previously (Wu et al., 2006). Briefly, lipid ingredients, including egg yolk phosphatidylcholine, cholesterol and Rg3, were dissolved in methanol and dried on a rotary evaporation in a round-bottom flask. The lipid film was further dried under vacuum and then hydrated with phosphate-buffered saline (PBS)

Table 1 Levels of factors used in BBD. Factors

Code

Ratio of lipid to drug (w/w) (A) Ratio of ePC to cholesterol (w/w) (B) Lipid concentration mg/ml (C)

X1 X2 X3

Range and levels −1

0

1

5 3 30

10 4 45

15 5 60

at room temperature (20 mM; pH 7.4). The lipid suspension was then extruded 3 times each through 0.2 ␮m and then 0.1 ␮m pore size polycarbonate membranes on a lipid extruder from Northern Lipids Inc. (Burnady, BC, Canada) driven by high pressure nitrogen. Encapsulation efficiency is one of the most important evaluation parameters for liposomes carriers (Yang et al., 2012), based on a single-factor test, three parameters that have significant effect on encapsulation efficiency were selected to investigate, they were the lipid to drug (w/w) (A), ratio of ePC to cholesterol (w/w) (B), and total lipid concentration (mg/ml) (C), were selected to optimize the composition of L-Rg3. The selected factors were subjected to analysis by the response surface methodology (RSM) with a three factor–three coded level Box–Behnken design (BBD). The range and the values of experiment variable investigated in this study are presented in Table 1. In addition, several confirmatory experiments were done according to the optimal composition. The relationship between each factor and index was fitted using data processing software Design-Expert trial version 8.0.6 (Stat-Ease Inc., Minneapolis). Regression coefficients and constants were calculated. In addition, accuracy of regression formula obtained was evaluated by fitness and correlation coefficient. Response surfaces that described the relationship between each factor and index were drawn according to fitting equation. Finally, we selected optimization formula from the response surface drawing to produce optimized L-Rg3 and executed predictive analysis. 2.4. Cytotoxicity of L-Rg3 Cytotoxicity of L-Rg3 was determined by the MTT assay (Zhai et al., 2008). Basically, A549 and HepG2 cells were transferred to 96-well tissue culture plates at 5 × 103 cells per well 24 h prior to drug treatment. The culture medium was then replaced with 200 ␮l of medium containing serial dilutions of L-Rg3 or Rg3 dissolved in dimethylsulfoxide (DMSO, the final concentration in medium less than 0.1%). Following 24 h incubation at 37 ◦ C, the cells were washed twice with PBS and cultured in fresh medium for an additional 48 h. A total of 20 ␮l MTT stock solution (5 mg/ml) was added to each well and the plates were incubated for 4 h at 37 ◦ C. Medium was then removed and DMSO was added to dissolve the blue formazan crystals converted from MTT. Cell viability was assessed by absorbance at 570 nm measured on a Biorad microplate reader. 2.5. Pharmacokinetics and biodistribution Thirty Wistar rats were divided into six groups of 5 animals each. The rats in each group were given Rg3 solution or L-Rg3 i.v. at a dose of 0.25, 0.5 or 1 mg/kg. Blood samples (∼500 ␮l) were collected at 0.033, 0.16, 0.5, 1, 2, 3, 5, 8 and 12 h after administration. Blood samples were placed in heparinized tubes, immediately centrifuged, and stored at −20 ◦ C until analysis. The plasma concentration of Rg3 was assayed by an LC–MS method after the samples were pretreated by a liquid–liquid extraction method. Briefly, to 100 ␮l plasma sample, 100 ␮l of methanol (containing Ginsenoside Rh2 as internal standard, 25 ng/ml) and 500 ␮l water were added. Samples were then vortex-mixed for 3 min and extracted with 2.0 ml ethyl acetate by vortex-mixing for 5 min. After centrifugation at 4500 × g for 10 min, the upper organic layer was transferred to another tube

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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and evaporated to dryness at 45 ◦ C under a gentle stream of nitrogen. The residue was dissolved in 200 ␮l of the mobile phase, and vortex-mixed for 1 min. A 20 ␮l aliquot of the solution was injected onto the LC/MS/MS system for analysis. The reasons we used a rat model for pharmacodynamics studies are as follows. First, we could use a large animal number in order to minimize the impact of individual differences. Second, using mice is difficult to collect enough samples for the detection. Third, normal animal is more useful as a model for the further investigate of pharmacokinetics and biodistribution of human (Andes, 2004; Mager et al., 2009). Twenty-four Wistar rats were randomly divided into two groups of 12 rats. The rats were administered Rg3 solution or L-Rg3 i.v. at a dose of 0.5 mg/kg. The animals were sacrificed, and selected tissues, including plasma, heart, liver, spleen, lung and kidney, were collected at 0.5, 2, 6 and 12 h after drug administration. Ice-cold physiological saline was used to rinse the tissues. Then the tissues were weighed accurately, cut into slices and homogenized in a ground glass tissue grinder after adding the appropriate amount of methanol (3 ml/g tissue). The uniform homogenates were immediately stored at −20 ◦ C until analysis. Tissue homogenates were pretreated and assayed according to the same procedure as those of plasma samples.

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2.6. Animal model

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1 × 107 /ml,

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A549 cells were adjusted to a concentration of and 0.2 ml cell suspensions were injected subcutaneously into the armpit of right anterior superior limbs of the mice. When the tumors reached palpable sizes, the mice were randomized into the following five groups (5 mice per group): a control group injected daily i.p. with saline; a Rg3 solution group, which received Rg3 solution by daily i.p. (injection at 1 mg/kg, dissolve in alcohol) for 21 days (QDx21); 3 L-Rg3 groups, which received daily i.p. injection and dosing levels of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg for 21 days (QDx21).

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2.7. In vivo anti-tumor activity test

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The length and width of tumor were callipered every 4 days. Throughout the experiments, the tumor size and body weight of the mice were measured. The longest tumor diameter (length a) and the diameters crossing the longest diameters at right angles (widths b) were measured with a slide caliper, then the tumor volumes (V) were calculated according to the following equation: V (mm3 ) = a (mm) × b (mm) × b (mm)/2 (Omokawa et al., 2010). During the experiment period, side effects including weight loss, change in behavior and dietary, response to stimulation, ruffling of fur and psychosis (distress) were monitored. When a mouse died or met an early removal criterion and sacrificed, the size of tumor and the number of days of survival were recorded. Inhibition of tumor was calculated using the formula: inhibition of tumor (%) = (1 − average tumor volume in treated group/average tumor volume in control) × 100. 2.8. Detection of microvessel density (MVD) Microvessel density was assessed by immunohistochemical analysis with antibodies to the endothelial marker CD34 and determined according to the method of Foote et al. (2005). In short, the hot spot of the immunostained sections was identified through initially screen at low magnifications (40× and 100×). There were the areas of highest neovascularization. Endothelial cell clusters that were yellow brown stained and clearly separate from adjacent tumor cells and other connective tissue elements were considered a single, countable microvessel. Within the hot spot area, the stained

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Table 2 Experimental design for three factors and experimental values of objective variables. Std

Run

11 9 2 1 12 6 13 7 16 14 15 4 17 10 5 3 8

Levels of independent factors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

X1

X2

X3

10.00 10.00 15.00 5.00 10.00 15.00 10.00 5.00 10.00 10.00 10.00 15.00 10.00 10.00 5.00 5.00 15.00

3.00 3.00 3.00 3.00 5.00 4.00 4.00 4.00 4.00 4.00 4.00 5.00 4.00 5.00 4.00 5.00 4.00

60.00 30.00 45.00 45.00 60.00 30.00 45.00 60.00 45.00 45.00 45.00 45.00 45.00 30.00 30.00 45.00 60.00

Response EE (%)

79.56 55.69 60.44 68.55 83.84 50.69 82.29 79.95 85.88 86.9 87.11 58.81 83.28 63.85 57.53 67.42 69.73

microvessels were counted in a single high-power (200×) field, and the average vessel count in 3 hot spots was considered the value of MVD. All counts were performed by three investigators in a blinded manner. Microvessel counts were compared between the observers and discrepant results were reassessed. The consensus was used as the final score for analysis. 2.9. Statistical analysis

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Statistical analysis was performed using ANOVA. The significance was designated at p < 0.05.

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3. Results

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3.1. Preparation of L-Rg3

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There were 17 experimental runs for optimizing the three individual parameters in the Box–Behnken design (BBD), and the experimental conditions and the encapsulation efficiency (EE) of Rg3 according to the factorial design is shown in Table 2. The results showed that the EE ranged from 50.69 to 87.11%. The statistical significance of the regression model was checked by F-test and pvalue, and the analysis of variance (ANOVA) for the response surface quadratic model is shown in Table 3, in which the small p-value for the model (<0.0001) implied the model was significant, p-value of less than 0.05 suggested model term was significant. The p-value for the ‘lack of fit’ test was 0.2472, indicating the quadratic model was adequate. By statistically processed and fitting, multiple second-order equations were obtained as follows: Final equation in terms of coded factors: R = + 85.09 − 4.22A + 1.21B + 10.67C − 0.13AB − 0.85AC 2

2

− 0.97BC − 13.77A − 7.51B − 6.84C

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2

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Final equation in terms of actual factors:

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R = − 197.90950 + 10.78130A + 64.47800B + 3.81973C

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2

− 0.025000AB − 0.011267AC − 0.064667BC − 0.55094A 2

− 7.51350B − 0.030416C

2

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The analysis of fitting result is shown in Table 4.

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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Table 3 Statistical analysis of variance for the experimental results. Source Model A B C AB AC BC A2 B2 C2 Residual Lack of fit Pure error Cor. total

264 265 266 267 268 269 270 271 272 273 274 275 276 277

Sum of squares 2429.14 142.64 11.71 909.94 0.063 2.86 3.76 798.78 237.70 197.19 48.71 29.61 19.10 2477.86

df

Mean squares

F value

p value Prob > F

9 1 1 1 1 1 1 1 1 1 7 3 4 16

269.90 142.64 11.71 909.94 0.063 2.86 3.76 798.78 237.70 197.19 6.96 9.87 4.77

38.79 20.50 1.68 130.76 0.008982 0.41 0.54 114.79 34.16 28.34

<0.0001 0.0027 0.2356 <0.0001 0.9272 0.5421 0.4860 <0.0001 0.0006 0.0011

The above regression equations quantitatively described the effects of three independent variables (A–C) on index and their correlation. The adjusted R2 for the predictive model 0.9551, as well as the statistical test results of equation parameters (Table 4), revealed that the experimental results adequately fitted the equation selected. It can be predicted to obtain response value of a random formula within the range of designed factor and level by regression equations. To better comprehend the predictive models of the results, three-dimensional graphs of the models, the response surface diagrams of encapsulation efficiency of L-Rg3 are shown in Fig. 1. With an increase of ratio of lipid to drug and ratio of ePC to cholesterol encapsulation efficiency ascended at first and descended at last as Fig. 1A and B shown. As shown in Fig. 1C, with an increase in lipid

2.07

0.2472

Significant

Not significant

Table 4 The results of fitting second-order equations. Std. dev. Mean C.V. % PRESS

2.64 71.85 3.67 503.67

R-squared Adj R-squared Pred R-squared Adeq precision

0.9803 0.9551 0.7967 17.131

concentration, encapsulation efficiency ascended, and the factor was most significant (p < 0.0001). The suitability of the model equation for predicting the optimum response values was tested using the recommended optimum conditions. The set of optimum conditions, determined using the RSM optimization approach, were tested experimentally according to the model equation. Three batches of liposomes were prepared

Fig. 1. Response surface plot showing the effect of (A) ratio of lipid to drug, (B) ratio of ePC to cholesterol, and (C) lipid concentration.

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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according to the optimized formulation. Then EE of each batch were determined. The mean experimental EE 82.47% was close to the predicted results 89.69% (lipid concentration 56.82 mg/ml; ratio of lipid to drug 9.11, and ratio of ePC to cholesterol 4.03). L-Rg3 of optimized formulation was used for determination of particle size distribution and -potential. The particle size of L-Rg3 was 133.9 nm with a relatively narrow particle size. Value of potential for L-Rg3 was −23 mV, which showed that liposomes obtained had sufficient charge to inhibit aggregation of vesicles (Xiong et al., 2009).

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3.2. Cytotoxicity of L-Rg3

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Proliferation of A549 and HepG-2 cells were both inhibited in concentration-dependent manners. IC50 of Rg3 solution and L-Rg3 for A549 cell were respectively 126.43 ␮g/ml and 84.68 ␮g/ml; IC50 of Rg3 solution and L-Rg3 for HepG-2 cell were respectively 99.74 ␮g/ml and 65.87 ␮g/ml. No cytotoxicity of the unloaded liposome was observed in either cell-line studied. In both cells, the cytotoxicity of liposomes encapsulated with Rg3 was greater than that of the free drug. In addition, the cell killing effect was concentration-dependent, as shown in Fig. 2.

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3.3. Pharmacokinetics and bio-distribution

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The next step was to assess the pharmacokinetic behavior of Rg3 encapsulated in liposomes in Wistar rats after i.v. administration. The plasma concentration vs. time of Rg3 obtained after the injection is shown in Fig. 3. After injection of L-Rg3, the Rg3 concentration was still measurable after 12 h (shown in Fig. 3), while the drug injected free in Rg3 solution was not detectable even after 5 h. The comparative pharmacokinetic parameters after i.v. administration of the Rg3 formulations are shown in Table 5. The liposomes enhanced the Cmax and AUC of Rg3 by 1.19- and 1.52-fold, respectively (p < 0.05). Tissue distributions after administration of Rg3 in solution or incorporated in liposomes are presented in Fig. 4. Rg3 was extensively distributed in all tissues assayed. The L-Rg3 enhanced the concentration of Rg3 in all the tested tissues. Compared to Rg3 solution, L-Rg3 significantly enhanced the liver and lung distribution of the drug (p < 0.05).

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3.4. In vivo antitumor activity

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Tumor volume, inhibitive and survival rates were determined. The treatment began when the tumors were palpable after the mice were transplanted with tumor cells. As Fig. 5 shows, treatment with Rg3 solution and L-Rg3 both showed decrease in tumor volume compared with that of the controls during treatment period

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Fig. 2. Cytotoxicity of Rg3 solution and L-Rg3.

(control group, 1335 mm3 , p < 0.05). In addition, at the same concentration, inhibitive rate of tumor in the L-Rg3 group was higher Rg3 solution group (p < 0.05), shown in Table 6. At 21 days after the treatments, the mice in the control, L-Rg3, and Rg3 solution group were all alive. There were no significant abnormalities in psychosis, status of activity, reaction to stimulation, appetite or depilation of mice in the Rg3 solution, control or L-Rg3 group. However, the mice

Fig. 3. Plasma concentrations of Rg3 after i.v. administration of the drug solution and liposome formulations (n = 5).

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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Table 5 Pharmacokinetic parameters of Rg3 in plasma after i.v. administration (n = 5). Parameter

Rg3 solution (0.5 mg/kg)

AUC(0–t) (␮g/l h) AUC(0–∞) (␮g/l h) Cmax MRT(0–t) (h) MRT(0–∞) (h) t1/2 (h) CL (l/h/kg)

382.311 384.02 2185 0.18 0.196 0.54 52.081

*

L-Rg3 (0.5 mg/kg) 57.3 56.7 480.07 0.08 0.07 0.06 14.57

128.03* 118.37* 722.09* 0.06 0.07 0.08 8.56

581.986 583.676 3343.05 0.174 0.184 0.491 34.266

p < 0.05 when compared with Rg3 group.

Table 6 The effect of various Rg3 formulations on the BALB/c nude mice (n = 5). Formulation

Normal saline Rg3 solution (1 mg/kg) L-Rg3 (0.3 mg/kg) L-Rg3 (1.0 mg/kg) L-Rg3 (3.0 mg/kg)

Number of mice

Body weight (g)

Begin

End

Beginning

5 5 5 5 5

5 5 5 5 5

20.17 20.35 21.61 20.40 21.28

± ± ± ± ±

1.32 0.44 1.37 1.05 1.34

Inhibition (%) End 17.81 21.41 22.55 22.33 22.76

± ± ± ± ±

1.27 0.85 0.91 0.76 0.82

None 50.86 48.03 59.16 64.98

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relative to the L-Rg3 and Rg3 solution group, the control group lost more weight, shown in Fig. 6.

its anti-angiogenic effect was further improved when delivery by liposomes.

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3.5. Detection of microvessel density (MVD)

4. Discussion

The result of MVD is shown in Figs. 7 and 8. Compared with the control group, MVD value in the Rg3 solution and L-Rg3 group decreased significantly, especially in the L-Rg3 group (p < 0.05) (Fig. 8). In addition, at the same concentration, MVD of L-Rg3 group was small than Rg3 solution group (p < 0.05). The results indicated that Ginsenoside Rg3 inhibited tumor angiogenesis and

Despite their high antitumor efficacy of chemotherapy drugs such as doxorubicin, the use of chemotherapy agents is limited due to the different toxicity including testicular and myocardial damage. It is necessary to develop highly efficient anticancer agents or adjuncts to reduce or minimize adverse effects for the advanced therapy.

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Fig. 4. Tissue distribution of Rg3 after i.v. administration. After i.v. (A) 0.5 h, (B) 2 h, (C) 6 h, and (D) 12 h (n = 3).

Please cite this article in press as: Yu, H., et al., Development of liposomal Ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.065

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Fig. 5. Tumor volume growth curves (n = 5). Fig. 8. MVD of different group (n = 5).

Fig. 6. The mice weight change curve (n = 5).

Anti-angiogenic therapy for cancer is aimed to prevent tumor cell proliferation and tumor expansion by inhibiting tumor-related angiogenesis, thus depriving the tumors of essential nutrients and oxygen (Abe et al., 2007; Folkman, 1971; Shpitz et al., 2003). Recently Rg3 has been suggested to inhibit cancer cell growth, invasion and metastasis, e.g. lung carcinoma (Lu et al., 2008), prostate cancer (Kim et al., 2004), colorectal cancer (Luo et al., 2008), ovarian cancer (Xu et al., 2007, 2008) and breast cancer (Zhang et al., 2008). Studies have demonstrated oral Rg3 with fairly low or undetectable plasma concentrations being achieved (Wang et al., 1999). Research in this area has been focusing on the development of alternative formulations to improve drug delivery either by oral or IV route. In this study, L-Rg3 was designed and evaluated for use as a delivery agent. Response surface methodology (RSM) is a collection of mathematical and statistical technique which quantifies the functional relationship between a number of measured response variables and several explanatory factors, hereby to acquire an optimal response by using a sequence of tests (Hatambeygi et al., 2011). Based on the preliminary experiments and our previous studies, three factors (ratio of lipid to drug, ratio of ePC to cholesterol and lipid concentration) were identified as key factors responsible for

Q3 Fig. 7. Immunohistochemical analysis of tumor MVD changes. The blood vessels in tumor tissues were stained in yellow brown. (A) Control group; (B) Rg3 solution group; (C) L-Rg3 (0.3 mg/kg) group; (D) L-Rg3 (1.0 mg/kg) group; and (E) L-Rg3 (3.0 mg/kg). Scale bars: 100 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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EE. Ratio of lipid to drug was chosen because Rg3 concentration is very important for EE. EE will increase with Rg3 concentration augmentation. But when Rg3 concentration augmentation reaches the pole, Rg3 will leak from liposome and the EE will decrease due to the excessive accumulation of Rg3 in the lipid bilayer. Ratio of ePC to cholesterol was another important factor because cholesterol, as one of the ingredients of membrane, acts as a stabilizer of the membrane to increase EE and reduce the drug leakage (Yingjie, 2007). High total lipid contents used in preparation would result in more lipid carrier formation. The encapsulation capacity of drugs affecting the quality of the liposomes, while high encapsulation efficiency is basic factor in determining clinical drug effects. The optimal preparation condition for Rg3-liposome by response surface methodology was as follows: lipid concentration56.82 mg/ml; ratio of lipid to drug 9.11, ratio lipid to cholesterol 4.03. Under these conditions, the experimental encapsulation efficiency of Rg3 was 82.47 ± 0.74%. We compared the antitumor activity of Rg3 loaded liposomes with free drug in two human cancer cell line. The liposomes could enhance the cytotoxicity in both cell lines. IC50 of Rg3 solution and L-Rg3 to A549 cell were respectively 126.43 ␮g/ml and 84.68 ␮g/ml; IC50 of Rg3 solution and L-Rg3 to Hepg-2 cell were respectively 99.74 ␮g/ml and 65.87 ␮g/ml. Importantly, blank liposomes did not have any apparent effect on the proliferation of A549 and HepG-2 cells in the whole range of concentrations examined during the treatment with Rg3 formulations, confirming that anticancer activity was due to uptake ability of the drugs, rather than the carrier system. The blood Rg3 concentration–time profiles in rats after i.v. injection of drug, in either a free drug and liposome shown that encapsulation of Rg3 in liposome showed marked differences in terms of the pharmacokinetic parameters calculated for free Rg3. In particular, the values of Cmax and AUC were found to be much higher for Rg3 vectored by liposomes. The liposomes enhanced the Cmax and AUC of Rg3 by 1.19-fold and 1.52-fold (p < 0.05). After 12 h, liposomes were still retained in the plasma, whereas free drug had almost disappeared from circulation after 5 h. The carrier system improved the relative bioavailability of the drug. The biodistribution studies with liposomes indicated that after i.v. injection there was a considerable increase in drug uptake through enhanced permeation and retention (EPR) (lung and liver, p < 0.05). Most solid tumors are known to exhibit highly enhanced vascular permeability, will result in the permeability of the liposomes in tumor tissue enhancement, and enhance the anti-tumor potency of drug (Maeda et al., 2003). The tumor growth rate in mice treated with Rg3 solution and L-Rg3showed in Fig. 4 indicated significant antitumor effect of liposome. After 21 days treated, the average tumor volume in all group had increased relatively slowly and attained about 607 mm3 (L-Rg3, 0.3 mg/kg), 477 mm3 (L-Rg3, 1.0 mg/kg), 409 mm3 (L-Rg3, 3.0 mg/kg) and 574 mm3 (Rg3 solution, 1.0 mg/kg). In the same concentration, Rg3 loaded liposome showed a significantly higher antitumor efficacy than did free drug. Angiogenesis plays an important role in both tumor growth and metastasis (Folkman, 1990). Angiogenesis is tightly regulated by pro-angiogenic and anti-endothelial growth factors. MVD is accepted as a standard indicator of angiogenesis (Magnon et al., 2007). In the present study, MVD in the Rg3 solution and L-Rg3 group decreased obviously, especially in the L-Rg3 group. Our data indicated that liposome reinforced inhibitory effect of angiogenesis by decreasing MVD value.

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We successfully obtained liposome containing Rg3, the preparation conditions were optimized by response surface. The present

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