Nanostructured lipid carriers as novel carrier for parenteral delivery of docetaxel

Colloids and Surfaces B: Biointerfaces 85 (2011) 262–269

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Nanostructured lipid carriers as novel carrier for parenteral delivery of docetaxel Donghua Liu, Zhihong Liu, Lili Wang, Cai Zhang, Na Zhang ∗ School of Pharmaceutical Science, Shandong University, 44 Wenhua Xi Road, Ji’nan 250012, China

a r t i c l e

i n f o

Article history: Received 23 November 2010 Received in revised form 25 February 2011 Accepted 25 February 2011 Available online 8 March 2011 Keywords: Docetaxel Nanostructured lipid carriers (NLC) Cancer therapy Cytotoxicity

a b s t r a c t The aim of this study was to design docetaxel-loaded nanostructured lipid carriers (DTX-NLC) to reduce toxicity and improve therapeutic efficacy. Docetaxel-loaded nanostructured lipid carriers (DTX-NLC) were prepared by the modified film ultrasonication–dispersion method. The DTX-NLC were characterized by particle size distribution, zeta potential and entrapment efficiency. In vitro cytotoxicity of DTX-NLC was evaluated by MTT assay against three human cancer cell lines and one murine malignant melanoma (B16). AnnexinV-FITC kit was used to measure the percentage of apoptosis induced by Duopafei® or DTX-NLC. In vivo anti-tumor efficacy was evaluated in Kunming mice bearing murine malignant melanoma (B16). Compared with Duopafei® , DTX-NLC revealed more cytotoxicity against A549 cells by inducing more apoptosis and more G2/M arrest. The inhibition rates of Duopafei® , DTX-NLC (10 mg/kg) and DTX-NLC (20 mg/kg) were 42.74%, 62.69% and 90.36%, respectively, indicating that DTX-NLC could more effectively inhibit tumor growth. The results of the body weight variations of mice also showed that compared with Duopafei® , DTX-NLC had lower toxicity during the therapeutic procedure. These results suggest that DTX-NLC may be a promising drug delivery system for cancer therapy. To our knowledge, this was the first report about DTX-NLC for murine malignant melanoma treatment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Recently studies indicated that nanosized drug delivery systems can enhance efficacy and reduce side effects of cytotoxic drug [1]. Several nanosystems have been developed and among these, the albumin–paclitaxel nanoparticles were approved in early 2005 in the chemotherapy for metastatic breast cancer [2]. Lipid nanoparticles represent drug vehicles composed of physiological lipids such as phospholipids, cholesterol, cholesterolesters and triglycerides [3]. The biological origin of the nanocarrier material offers a number of advantages making lipid nanoparticles one of the ideal drug delivery vehicles [4]. The bioacceptable and biodegradable nature of these systems makes them less toxic as compared to other nanocarriers (e.g. polymeric nanoparticles). The nanostructured lipid carriers (NLC) are presented as an improved generation of lipid nanoparticles [5,6], which is developed from solid lipid nanoparticle (SLN) system. It consists of solid lipid matrices with spatially incompatible liquid lipids, results in a structure with more imperfections in crystal to accommodate the drug, and thus gets a higher drug loading capacity. NLC system shares advantages of SLN, e.g. controlled drug release, bio-

∗ Corresponding author. Tel.: +86 531 88382015; fax: +86 531 88382548. E-mail address: [email protected] (N. Zhang). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.02.038

compatibility and the possibility of production on large industrial scale. Furthermore, it minimizes or avoids some potential problems associated with SLN, such as drug leakage during storage and limitation in drug loading capacity. Therefore, NLC are promising carrier to increase the drug loading efficiency and prolong the half-life of DTX. It can prolong exposure of tumor cells to antitumor drug, enhance permeability and retention (EPR) effect [7], and subsequently increase the therapeutic effect of anti-tumor drug. Docetaxel (DTX) is a semisynthetic taxoid derived from the European yew tree, Taxus baccata, and it is a mitotic spindle poison that accelerates the microtubule assembly from tubulin and blocks the depolymerization of the microtubule [8]. It has demonstrated extraordinary anticancer effects both in vitro and in vivo against a variety of tumors including lung, ovaries, breast, leukemia, malignant melanoma, etc. [9–11]. DTX like other taxanes such as paclitaxel shows low water solubility, hence Taxotere® and Duopafei® consist of high concentrations of Tween 80. Duopafei® was docetaxel injection which was provided by Qilu Pharmaceutical Co., Ltd. in China. The formulation compositions and the clinical application of Duopafei® were both similar to Taxotere® . Taxotere® has been associated with severe hypersensitivity reactions due to the side effects of Tween 80 [12]. Therefore, developing a suitable drug delivery system without Tween 80 is meaningful.

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In order to eliminate the side effects of the Tween 80-based formulation and in an attempt to increase the drug solubility, alternative nanocarriers have been suggested, including polymeric nanoparticles [13,14], liposomes [15] and SLN [16]. These nanocarriers have shown potential in improving efficacy and minimizing side effects of Taxotere® . While, the major problems associated with these nanocarriers are the poor stability, unsatisfactory batch to batch reproducibility, difficulty in sterilization, and low drug loading capacity [17]. NLC system can share advantages of them and minimize or avoid their potential problems [18]. To our knowledge, previous studies have reported that DTX-NLC have an organtargeting effect and have potential for the treatment of lung cancer. However, no report on the efficacy evaluation of NLC formulation of DTX against malignant melanoma was identified. So this was the first report about DTX-NLC for murine malignant melanoma treatment. In this work, DTX loaded NLC (DTX-NLC) were designed, which mainly consisted of biodegradable and biocompatible components such as stearic acid, glyceryl monostearate, soya lecithin and oleic acid. The in vitro cytotoxicity of Duopafei® and DTX-NLC against three human cancer cell lines and one murine malignant melanoma (B16) were evaluated. The apoptosis and cell cycle analysis was assessed by flow cytometry to investigate the mechanisms of action of Duopafei® and DTX-NLC on cancer cell. DTX has extraordinary anticancer effects against malignant melanoma and B16 are murine malignant melanoma cells, which are sensitive to growth inhibition of DTX. So, in vivo anti-tumor efficacy was evaluated in Kunming mice bearing B16. The results of in vitro and in vivo studies showed that DTX-NLC could reduce side effects and improve efficacy of Duopafei® . 2. Materials and methods 2.1. Materials Injectable soya lecithin (phosphatidylcholine accounts for 95%, pH = 5.0–7.0) was provided by Shanghai Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Pluronic F68 (F68) was purchased from Sigma (China). Stearic acid, glyceryl monostearate and oleic acid were purchased from Shanghai Chineway Pharm. Tech. Co., Ltd. (Shanghai, China). Duopafei® was provided by Qilu Pharmaceutical Co., Ltd. (Jinan, China). AnnexinV-FITC kit and Hoechst 33342 were purchased from Jingmei Biotech. Co., Ltd. (Jinan, China). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially. Human hepatocellular liver carcinoma (HepG2), ovary cancer cells (SKOV3), lung adenocarcinoma (A549) and murine malignant melanoma (B16) cell line were obtained from Shandong Institute of Immunopharmacology and Immunotherapy (Shandong, China). Human umbilical vein endothelial cells (HUVEC) were purchased from Sciencell (China). 2.2. Preparation of DTX-loaded nanostructured lipid carriers DTX-NLC were prepared by the modified film ultrasonication– dispersion method. The desired amounts of stearic acid, glyceryl monostearate, soya lecithin, oleic acid and DTX were dissolved in ethanol. Organic solvent was evaporated to form oil phase (RE5298, Shanghai Yarong Biochemistry Instrument Factory, China) under vacuum in water bath at 50 ◦ C for 30 min. At the same time, the desired amounts of soya lecithin and F68 were dissolved in distilled water to form aqueous phase. The oil phase and aqueous phase were kept in 80 ◦ C water bath. The aqueous phase was added to oil phase and the mixture was stirred for 5 min under this temperature. After water-bath ultrasonic treatment (HS-3120,

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Tianjin Hengao Technology Development Co. Ltd., China), DTXNLC were obtained by solidification in ice bath. Then DTX-NLC were lyophilized (LGJ0.5, Beijing Four-Ring Scientific Instrument Co., China). 2.3. Determination of entrapment efficiency and drug loading The desired amounts of DTX-NLC (0.1 mL) were dispersed in 2.9 mL of 0.5 wt% Tween 80 phosphate buffer solution (PBS, pH 7.4) and shaken (XW-80A, Instruments factory of Shanghai Medical University, China) for 3 min to dissolve the free drugs [19]. The resulting dispersions were centrifuged for 10 min at 25,000 rpm (3K30, Sigma, Germany). The drug content in the supernatant after centrifugation was measured by HPLC method using an SPD-10Avp Shimadzu pump and LC-10Avp Shimadzu UV–vis detector. Samples were determined by an HPLC method with the following conditions: Venusil XBP C-18 (4.6 mm × 250 mm, pore size 5 ␮m, Agela), the mobile phase: acetonitrile:water (55:45, v/v), flow rate: 1.0 mL/min, and measured wavelength: 230 nm. The original DTXNLC (0.01 mL) were dissolved in 0.49 mL methanol and the drug content in the original DTX-NLC were detected by the same HPLC method described above. The calibration curve of peak area against concentration of DTX was A = 12,684C − 722.76 (R = 0.9998) under the concentration of DTX 1–50 ␮g/mL (r = 0.9998, where A = peak area and C = DTX concentration) and the limit of quantitation was 0.02 ␮g/mL. The drug entrapment efficiency (EE) and drug loading (DL) of DTX-NLC were calculated from Eqs. (1) and (2) EE% =

Wtotal − Wfree × 100 Wtotal

(1)

DL% =

Wtotal − Wfree × 100 Wlipids

(2)

Wtotal , Wfree and Wlipids are the weight of drug added in system, analyzed weight of drug in supernatant and weight of lipid added in system, respectively. 2.4. Characterization of NLC The morphology of DTX-NLC was examined by transmission electron microscopy (JEM-1200EX, Japan). Samples were prepared by placing a drop of fresh prepared NLC suspension onto a copper grid and air-dried, following negative staining with a drop of 3% aqueous solution of sodium phosphotungstate for contrast enhancement. The average diameter and polydispersity index were determined by laser light scattering (Zetasizer 3000SH, Malvern Instruments Ltd., England). Zeta potential was measured by the laser Doppler anemometry (LDA) on ZetaPlus Zeta Potential Analyzer (Brookheaven Instruments Corporation). The lyophilized DTX-NLC were suspended with PBS (pH 7.4) before measured. All measurements were performed at 25 ◦ C. Calculation of the size and polydispersity indices was achieved using the software provided by the manufacturer. The diameters mean value was calculated from the measurements performed at least in triplicates. 2.5. In vitro release of DTX-loaded nanostructured lipid carriers In vitro release of DTX from the NLC and Duopafei® was evaluated using dialysis bag diffusion technique. Typically, 0.25 mL of DTX solution (1 mg/mL, Duopafei® diluted with deionized water) and 0.25 mL DTX-NLC (1 mg/mL, freeze-dried DTX-NLC suspended in de-ionized water) were placed into a pre-swelled dialysis bag with 8–12 kDa molecular weight cutoff. The bag was incubated in 15 mL release medium (0.5% of Tween 80 in PBS, pH 7.4) at 37 ◦ C under horizontal shaking [20]. At predetermined time points, the

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dialysis bag was taken out and replaced into a new container filling with 15 mL fresh medium. The amount of docetaxel released was determined by an HPLC method with the following conditions: Venusil XBP C-18 (4.6 mm × 250 mm, pore size 5 ␮m, Agela), the mobile phase: acetonitrile:water (65:35, v/v), flow rate: 1.0 mL/min, and measured wavelength: 230 nm. 2.6. In vitro cytotoxicity studies The cytotoxicity of DTX-NLC was tested in HepG2, SKOV3, A549 and B16 cells using the MTT assay [21]. Briefly, cells were seeded in a 96-well plate at a density of 4000 cells/well and allowed to adhere for 24 h prior to the assay. Cells were exposed to a series of doses of Duopafei® , blank NLC, or DTX-NLC, respectively, at 37 ◦ C. After 96 h of incubation, 20 ␮L of MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazoniumbromide) solution (5 mg/mL) was added to each well of the plate. After incubation for 4 h, 200 ␮L/well of DMSO was added to dissolve the contents in the plate, and the absorbance of the obtained DMSO solution was measured at 570 nm and 630 nm by a microplate reader (FL600, Bio-Tek Inc., Winooski, VT). All experiments were repeated in thrice. 2.7. Proliferation inhibition on HUVEC Proliferation inhibition of DTX-NLC was tested on human umbilical vein endothelial cells (HUVEC). HUVEC were seeded into 96-well plates and allowed to attach for 24 h. Cells were exposed to a series of doses of Duopafei® or DTX-NLC, respectively, at 37 ◦ C. Control group received culture media only. After incubation for 96 h, 20 ␮L of MTT solution (5 mg/mL) was added to each well of the plate. After incubating for 4 h, 200 ␮L/well of DMSO was added, and the absorbance of the solution was measured at 570 nm and 630 nm by a microplate reader (FL600, Bio-Tek Inc., Winooski, VT). All experiments were repeated three times.

Ministry of Health of the People’s Republic of China (document no. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. Mice implanted with B16 cells were used to qualify the efficacy of DTX-NLC administrated by intravenous injection. The mice were subcutaneously injected at the right axillary space with 0.1 mL of cell suspension containing 5 × 104 B16 cells [23]. Treatments were started after 8–10 days of implantation. The mice with tumor volume of ∼100 mm3 were selected and this day was designated as ‘Day 0’. The mice were randomly assigned to four treatment groups, with five mice in each group. Each group of mice was treated once a week by tail vein injection with the different formulations as described in the following: (A) physiological saline, as control group; (B) Duopafei® (dosage of 10 mg/kg, diluted in physiological saline); (C) DTX-NLC (dosage of 10 mg/kg); (D) DTX-NLC (dosage of 20 mg/kg). All mice were labeled, and tumors were measured every other day with calipers during the period of study. The tumor volume was calculated by the formula (W2 × L)/2, where W is the tumor measurement at the widest point and L the tumor dimension at the longest point. Each animal was weighed at the time of treatment, so that dosages could be adjusted to achieve the mg/kg amounts reported. Animals also were weighed every other day during the experiments period. After 20 days, the tumors were dislodged and weighed. The inhibition ratio (IR) could be defined as follows: inhibition ratio (%) = ((Wc − Wt)/Wc) × 100%. Wc and Wt stand for the average tumor weight for control group and treatment group, respectively [23]. 2.11. Statistical analysis Results were presented as mean ± SD. Statistical comparisons were made by t-test or ANOVA analysis. The accepted level of significance was P < 0.05.

2.8. Apoptosis determinations

3. Results and discussion

AnnexinV-FITC kit was used to measure the percentage of apoptosis induced by Duopafei® or DTX-NLC at dosages of 0.1, 2 or 10 ␮M. After treated for 24 h, cells were harvested and washed with PBS at 4 ◦ C and then resuspended in 100 ␮L binding buffer (1 × 106 cells/mL) containing 5 ␮L of AnnexinV-FITC and 10 ␮L of propidium iodide (PI). After incubated away from light for 10–15 min at room temperature, stained cells were analyzed by flow cytometry [22].

3.1. Preparation of DTX-loaded nanostructured lipid carriers

2.9. Cell cycle analysis Cell cycle analysis was assessed by flow cytometry. Briefly, cells (at least 1 × 106 cells) incubated with Duopafei® or DTX-NLC at dosage of 0.1 or 2 ␮M for 4 h, 8 h, 12 h and 24 h were harvested and washed with PBS, respectively. After fixed in 75% ethanol for 12 h at 4 ◦ C, these cells were washed twice and resuspended in RNase solution (100 ␮g/mL, 100 ␮L) and incubated for 30 min at 37 ◦ C. Then, 100 ␮L of PI (100 ␮g/mL) was added followed by another 30 min incubation at 37 ◦ C. Fluorescence was quantified on a flow cytometry, and the percentage of cells in each phase was calculated using ModFit software. 2.10. In vivo anti-tumor efficacy The Kunming mice (female) used in this study were purchased from the Medical Animal Test Center of Shandong University, 6–8 weeks old and weighing about 18–22 g. All experiments were carried out in compliance with the Animal Management Rules of the

The parameters such as weight ratio of drug to lipids (lecithin, stearic acid, glyceryl monostearate and oleic acid) (D/L ratios), weight ratio of solid lipids to liquid lipids (S/L ratios), the concentration of F68 (C) and the bath temperature (T) were optimized each at three or four levels taking the entrapment efficiency as index. When one factor was under investigated, the other three were fixed. The effects of the four influential factors, D/L ratios (X1), S/L ratios (X2), C (X3), T (X4) on encapsulation efficiency (EE) of DTXNLC are shown in Table 1. The results showed that the maximal value of EE could be reached when D/L ratio was set at 1:20 or 1:25 and S/L ratio was set at 3:1. The optimized concentration of F68 solution and the bath temperature were 1.5% and 50 ◦ C, respectively. The drug loading could be enhanced with the increase of D/L ratio, while a higher lipids ratio (the weight ratio of drug to lipids was 1:20 to 1:25) improved EE in this study. Moreover, EE value increased with the increase of S/L ratio, concentration of F68 solution and the bath temperature. Ultimately, each factor was set as three levels for L9 (34 ) orthogonal experiment. The D/L ratio was set at 1:10, 1:15 and 1:20 for gaining the higher drug loading. The value of EE was highest when S/L ratio was 3:1, so S/L ratio was set at 3:1, 4:1 and 9:1 for L9 (34 ) orthogonal experiment. The levels of the other three factors were set like Table 1. The results of L9 (34 ) orthogonal experiment of DTX-NLC are shown in Table 2. The optimal formulation was as follows according to Rj and K: D/L ratio, S/ratio, concentration of F68 and the bath temperature

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265

Table 1 The levels of experimental factors and the DTX-NLC entrapment efficiencies (n = 3).

1 2 3 4

X1 (w/w)

EE (%)

1:10 1:15 1:20 1:25

72.1 80.29 86.64 87.10

± ± ± ±

0.53 0.94 0.73 0.69

X2 (w/w)

EE (%)

1:1 3:1 4:1 9:1

80.89 89.30 89.14 84.00

± ± ± ±

1.01 0.95 1.21 0.86

X3 (w/v)

EE (%)

X4 (◦ C)

EE (%)

0.5% 1% 1.5%

80.18 ± 1.00 87.26 ± 1.32 88.21 ± 0.91

30 40 50

80.18 ± 0.76 84.15 ± 0.89 89.39 ± 099

X1 : weight ratio of drug to lipids (D/L ratios); X2 : weight ratio of solid lipids to liquid lipids (S/L ratios); X3 : concentration of F68 solution (C); X4 : the bath temperature (T). Table 2 The results of L9 (34 ) orthogonal experiment of DTX-NLC (n = 3). Factors

S/L ratios (w/w)

D/L ratio (w/w)

C (%)

T (◦ C)

EE (%)

1 2 3 4 5 6 7 8 9 K1 K2 K3 Rj

9:1 9:1 9:1 4:1 4:1 4:1 3:1 3:1 3:1 84.120 83.977 90.533 6.746

1:10 1:15 1:20 1:10 1:15 1:20 1:10 1:15 1:20 84.257 87.097 87.277 3.020

0.5 1 1.5 1 1.5 0.5 1.5 0.5 1 84.050 84.037 89.543 5.493

50 40 30 30 50 40 40 30 50 86.523 85.827 86.280 1.030

80.32 83.45 88.59 80.92 88.51 82.5 91.53 89.33 90.74

was 1:20, 3:1, 1.5% and 50 ◦ C, respectively. The optimized formulation was repeated in triplicates. 3.2. Entrapment efficiency and drug loading The average entrapment efficiency and the average drug loading of fresh-prepared DTX-NLC were 89.72 ± 0.89% and 4.47 ± 0.24%, respectively. After lyophilized, the average entrapment efficiency and the average drug loading of freeze-dried DTX-NLC were 88.9 ± 1.02% and 4.25 ± 0.35%, respectively. Liquid lipids were added in lipids of NLC and the solubility of DTX in NLC dispersion increased up to 1.0 mg/mL. This value is high enough to be used in clinical studies (<0.74 mg/mL). In order to determine entrapment efficiency of DTX-NLC, 0.5 wt% Tween 80 phosphate buffer solution (PBS, pH 7.4) was used to dissolve the free drugs due to low water solubility of DTX. The solubility of DTX in 0.5 wt% Tween 80 phosphate buffer solution (PBS, pH 7.4) at room temperature was 46.45 ± 0.31 ␮g/mL. DTX had a good linear relationship in 0.5 wt% Tween 80 phosphate buffer solution (PBS, pH 7.4), when its concentration was between 1 and 10 ␮g/mL (r = 0.9998, n = 5). The average recovery of three concentrations (1, 5 and 10 ␮g/mL) was between 99% and 101%, RSD < 1%.

± ± ± ± ± ± ± ± ±

0.67 0.75 1.02 0.56 0.95 0.59 0.89 0.65 0.67

ing equation: ln ln(1/(1 − Q/100)) = 0.8801 lnt − 2.3642, r = 0.9986. Fig. 2 shows the sustained release profile of DTX-NLC. It was obvious that DTX released much slower from DTX-NLC than from Duopafei® . In the first 24 h, 77% DTX of DTX-NLC was released and at about 96 h, almost all the loaded drug was released from DTXNLC. In contrast, the release of DTX from Duopafei® was fast and approximately 100% of the drug was released after incubated for 24 h. The difference between the release properties of DTX from Duopafei® and DTX-NLC is evidently attributed to the prolonged release function of NLC. Lipophilic DTX was held by the lipid core of the NLC and the drug released mainly through dissolution and diffusion. This result implied that DTX could be released slowly from DTX-NLC and could keep constant concentration for relative long period. Therefore, DTX-NLC might reduce

3.3. Characterization of DTX-loaded nanostructured lipid carriers The fresh-prepared DTX-NLC were spherical or ellipsoidal in shape (Fig. 1A). Particle diameters ranged from 100 to 300 nm with a mean diameter of 193.47 ± 5.69 nm and the zeta potential was −33.17 ± 1.20 mV. Freeze-dried DTX-NLC suspended in deionized water still were spherical or ellipsoidal in shape (Fig. 1B). Particle diameters ranged from 100 to 300 nm with a mean diameter of 203.67 ± 4.15 nm and −31.17 ± 2.20 mV. 3.4. In vitro drug release The release experiment was conducted under sink conditions and the dynamic dialysis was employed for separation of free drug from DTX-NLC. The release of DTX from DTX-NLC followed the Weibull equation and could be expressed by the follow-

Fig. 1. Transmission electron photomicrograms of DTX-NLC: (A) fresh-prepared DTX-NLC; (B) freeze-dried DTX-NLC.

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Accumulation release (%)

120

Table 4 IC50 of HUVEC incubated with Duopafei® and DTX-NLC (n = 3).

100

Cell line HUVEC

80

*

60

DTX-NLC

20

0

20

40

60

80

100

120

Time (h) Fig. 2. In vitro release profile of DTX from Duopafei® and DTX-NLC in phosphatebuffered saline (0.5% of Tween-80 in PBS, pH 7.4) at 37 ± 0.5 ◦ C (n = 3).

the frequency of administration. This is good for the clinical application.

3.5. In vitro cytotoxicity To assess the cytotoxicity of Duopafei® and DTX-NLC, their tumor-inhibiting activity was determined against three human cancer cell lines. The IC50 of Duopafei® and DTX-NLC for HepG2, SKOV3, A549 and B16 (n = 3), are presented in Table 3, respectively. Duopafei® and DTX-NLC showed a clear dose-dependent cytotoxicity against these cells with DTX at an equivalent dose from 0.01 to 10 ␮M. DTX-NLC have decreased the IC50 value for both cell lines and there was statistical significance in the IC50 values of DTX-NLC compared to Duopafei® , implying that DTX-NLC showed higher cytotoxicity against these cells. On the other hand, blank NLC had no significant effects on the cell growth (Table 3). Indeed, the differences in viability observed on cells that incubated with blank NLC and the non-treated cells were not statistically significant (P > 0.5). These negligible toxicity results observed for NLC, which are consistent with other reports [24] and could be explained by the low concentration of NLC present in the experimental conditions of the study. DTX-NLC showed higher cytotoxicity against cancer cells. The results are in accordance with previous studies that cytotoxicity of drug-loaded lipid based nanoparticles was higher than that of free drugs [18,20]. In many SLN formulations less than half of the loaded drug is released during the cell cytotoxicity test, but these formulations are sometimes more cytotoxic to the cancer cells than the corresponding free drug. In other words, the cytotoxic compounds that remain unreleased and associated with the lipid based nanoparticles also appear effective [25,26]. Possible mechanism underlying the enhanced efficacy of DTX-NLC against cancer cells is that lipid nanoparticles may carry drug into the cancer cells by endocytosis and enhance intracellular drug accumulation by nanoparticle uptake [27,28].

Table 3 IC50 of HepG2, SKOV3, A549 and B16 cells incubated with Duopafei® , DTX-NLC and blank-NLC at 96 h (n = 3). Cell line

Duopafei®

HepG2 A549 SK-OV-3 B16

0.96 0.74 0.08 0.72

* **

DTX-NLC

1.96 ± 0.16

1.67 ± 0.14*

®

P < 0.05 versus Duopafei .

Duopafei ®

40

0

Duopafei®

± ± ± ±

0.05 0.02 0.04 0.10

P < 0.05 versus Duopafei® . P < 0.01 versus Duopafei® .

DTX-NLC 0.47 0.15 0.02 0.44

± ± ± ±

0.03** 0.08** 0.01* 0.08*

Blank-NLC 30.26 17.50 10.11 26.34

± ± ± ±

0.1 0.09 0.11 0.3

3.6. Proliferation inhibition on HUVEC Considering the important role of the angiogenic vessels in tumor growth, the cytotoxicity of Duopafei® and DTX-NLC was investigated against blood vessel endothelial cells and human umbilical vein endothelial cells (HUVEC) was selected as the model cell. The IC50 of Duopafei® and DTX-NLC was calculated and shown in Table 4, which presented the in vitro inhibition of Duopafei® and DTX-NLC for HUVEC (n = 3). There was statistical significance in the IC50 values of DTX-NLC and Duopafei® , implying that compared with Duopafei® , DTX-NLC had greater sensitivity to HUVEC and could effectively inhibit the proliferation of tumor endothelial cells. DTX-NLC showed higher cytotoxicity against HUVEC and the results are in accordance with previous studies that cytotoxicity of DTX-NLC against cancer cells was higher than that of Duopafei® . These results showed that, besides cancer cells, NLC also may enhance intracellular drug accumulation in tumor blood vessel endothelial cells by nanoparticle uptake. These results indicated that the higher cytotoxicity against tumor blood vessel endothelial cells would enhance the tumor-inhibiting activity of DTX-NLC. 3.7. Apoptosis determinations and cell cycle analysis Docetaxel have been shown to target tubulin causing stabilization of microtubules, which results in cell-cycle arrest and apoptosis [18]. As a result, attention was given to the well-studied effects of docetaxel on tumor cells, such as cell cycle distribution and apoptosis. In the present work, cell apoptosis induced by DTX-NLC were first detected in A549 cells on the basis of apoptosis as the predominant mechanism of cell death in taxane chemotherapy [29]. The result showed that apoptosis occurred in cells treated with Duopafei® and DTX-NLC at the three different concentrations used in the experiments. To measure the apoptosis effect of DTX-NLC quantificationally, AnnexinV-FITC/PI was used to double stain the cells. AnnexinV-FITC staining combining with PI could distinguish early apoptosis from late apoptosis or living cells from dead cells [30]. In the flow cytometry quadrantal diagram, the lower left, lower right, upper right and upper left quadrants denoted viable, early apoptotic, late apoptotic and necrotic regions, respectively. From the flow cytometry profiles (Fig. 3), treated cells were found mostly in the upper right quadrant (Annexin-V positive), which indicated that Duopafei® and DTX-NLC could induce apoptosis in A549 cells. Cell apoptosis ratio increased significantly in a concentrationdependent manner. As shown in Fig. 3, the percentages of apoptotic cells after treatment with Duopafei® and DTX-NLC were 13.96 ± 0.91% and 16.73 ± 1.05% at dosage of 0.1 ␮M, 18.29 ± 0.56% and 23.67 ± 1.25% at dosage of 2 ␮M, and 31.3 ± 1.1% and 38.38 ± 0.93% at dosage of 10 ␮M, respectively, which indicated that DTX-NLC induced more apoptosis and produced higher cytotoxicity than Duopafei® . The percentage of apoptotic cells was obviously increased after the treatment with DTX-NLC and the better promotion was observed follow the treatment of the higher concentration. It has been reported that at the molecular level, docetaxel impairs mitosis and induces cell-cycle arrest [31]. Cell cycle was performed using a flow cytometry assay and the results are pre-

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Fig. 3. Induction of apoptosis on A549 cells by Duopafei® and DTX-NLC (n = 3). Apoptosis was evaluated after A549 cells treated with DTX-NLC or Duopafei® containing 0.1 ␮M, 2 ␮M and 10 ␮M drugs for 24 h, and then stained with Annexin-V-FITC and PI. (A) 0.1 ␮M; (B) 2 ␮M; (C) 10 ␮M. Flow cytometry profile represented Annexin-V-FITC staining in X axis and PI in Y axis. The early apoptotic cells were presented in the lower right quadrant, and the late apoptotic cells were presented in the upper right quadrant.

sented in Fig. 4. Compared with control cells, A549 cells treated with Duopafei® and DTX-NLC were accumulated predominantly in G2/M phases and fewer cells in G0/G1 phases. Fig. 4 shows a very obvious difference that DTX-NLC caused more cells arrest in the G2/M phase, compared with Duopafei® , which demonstrated that

DTX-NLC should have superior anti-tumor activity than Duopafei® . In addition, when A549 cells were treated with DTX-NLC or Duopafei® at concentration of 0.1 or 2 ␮M, respectively, the cells arrested in the G2/M phase increased with the incubation time from 4 to 24 h.

Fig. 4. Effects of treatment with Duopafei® or DTX-NLC for 4, 8, 12, 24 h on the cell cycle of A549 cells (n = 3). (A) Treated with equivalent docetaxel concentration of 0.1 ␮M; (B) treated with equivalent docetaxel concentration of 2 ␮M (*P < 0.05 versus Duopafei® ).

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Fig. 5. Antitumor effect of DTX-NLC. Data represent mean ± SD (n = 5). (A) Variation of tumor volume by intratumoral administration in B16 tumor-bearing mice. (B) Photographs of tumors from each treatment group excised on Day 20. (A) Saline control; (B) Duopafei® (10 mg/kg); (C) DTX-NLC (10 mg/kg); (D) DTX-NLC (20 mg/kg).

These results demonstrated that compared with Duopafei® , DTX-NLC could induce more apoptosis and more cells arrest in the G2/M phase, which is consistent with the data obtained by MTT analysis. The more uptake of DTX in DTX-NLC by tumor cells might be the reason. The results indicated that DTX-NLC should have superior anti-tumor activity. 3.8. In vivo anti-tumor effect The anti-tumor effect (in terms of tumor growth) is shown in Fig. 5. The obvious tumor regression was observed in mice treated with DTX-NLC. It was found that the tumor volumes treated with 10 mg/kg of DTX-NLC were smaller than those treated with the same dose of Duopafei® (P < 0.05) and the anti-tumor effect of DTXNLC (20 mg/kg) group was much stronger than that of DTX-NLC (10 mg/kg) group (P < 0.01). At the end of the test, tumor volume in mice treated with DTX-NLC (20 mg/kg) was 0.67 ± 0.65 cm3 , which were significantly smaller than the value of 5.76 ± 1.98 cm3 for Duopafei® group (P < 0.01). Fig. 6A shows the tumor weights of the three groups. The inhibition rates of Duopafei® , DTX-NLC (10 mg/kg) and DTX-NLC (20 mg/kg) were 42.74%, 62.69% and 90.36%, respectively. These results indicated that compared with Duopafei® , DTX-NLC showed more effective inhibition on tumor growth and the high dose of DTX-NLC showed much stronger antitumor effect. DTX-NLC delayed tumor development significantly than Duopafei® , which might result from the following reasons: (1)

The integrity structure of NLC containing liquid and solid lipid core could increase the solubility of docetaxel; (2) The optimal zeta potential and particle size of NLC could keep docetaxel stably entrapped in inner core of NLC and provide with convenience for accumulation and penetration into tumor area through the enhanced permeability and retention (EPR) effects [25,32]. (3) DTX can be slowly released from DTX-NLC due to the reservoir effect and kept at a constant concentration for long period in vivo. Major mechanism underlying the superiority of DTX-NLC against Duopafei® may be the continuous exposure of tumor mass to released DTX from the NLC. Therefore, the sustained release of DTX would be benefit to deliver its anti-tumor efficacy constantly. It has been reported that anti-tumor activity of chemotherapeutics depends on the dose and exposure time [33]. (4) DTX-NLC could effectively inhibit the proliferation of tumor blood vessel endothelial cells. It has been reported that destruction of tumor vascular system could have a profound impact on tumor growth, which involves the selective destruction of the tumor’s blood supply and tumor necrosis [33]. The accumulation and penetration of DTX-NLC into tumor area through the EPR effects would result in higher concentration of DTX-NLC in tumor tissue than that of DTX-NLC in normal tissue. Thus, this specific biodistribution of DTX-NLC would reduce the toxicity of DTX-NLC to normal cells. Fig. 6B shows the body weight variations of mice during the experiment period, no obvious body weight loss of the mice treated with DTX-NLC (10 mg/kg) was observed compared with those of the control group (P > 0.05). The weight loss induced by Duopafei® was much significant than those induced by DTX-NLC (10 mg/kg, P < 0.05) and DTX-NLC (20 mg/kg, P < 0.05). The analysis of body weight variations could be used to define the adverse effects of the different therapy regiments. These results lead to a conclusion that DTX-NLC generated less toxicity to mice than Duopafei® when administered intravenously under the present experiment condition and the high dose of DTX-NLC (20 mg/kg) also generated less toxicity to mice than Duopafei® (10 mg/kg), which will facilitate its future clinical application. Moreover, the mice receiving Duopafei® were observed in a weak state, whereas no obvious alteration was observed in the DTX-NLCtreated animals. Overall, these findings indicate that DTX-NLC (10 mg/kg) showed a little higher efficacy and lower side effects in murine malignant melanoma model when compared with Duopafei® (10 mg/kg). The high dose of DTX-NLC (20 mg/kg) showed much higher efficacy and lower side effects in murine malignant melanoma model. 4. Conclusions

Fig. 6. Tumor weight (A) and variation of body weight (B) by intratumoral administration in B16 tumor-bearing mice (n = 5).

In this delivery system, DTX can be well incorporated into NLC with high entrapment efficiency due to its lipophilicity. The in vitro release study showed a sustained and continuous release pattern of DTX-NLC. The MTT analysis proved DTX-NLC higher cytotoxicity

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against tumor cells and HUVEC. The in vitro studies demonstrated that compared with Duopafei® , DTX-NLC could induce more apoptosis and more cells arrest in the G2/M phase, indicating that DTX-NLC should have superior anti-tumor activity. In vivo evaluation further demonstrated the superior anticancer efficacy of DTX-NLC (10 mg/kg, 20 mg/kg) with relatively lower side effects compared with Duopafei® in an established B16 transplanted mice model. It was speculated that DTX-NLC decreased body weight loss by reducing in vivo toxicity in normal tissues. Thus, nanostructured lipid carriers possess advantages of reducing the high dose dependent toxicity of anticancer drugs and continuing research will definitely facilitate the current study. It is concluded that DTX-NLC had potential for the treatment of malignant melanoma. The patent of this research was applied for its potential clinical application. Funding The work was supported by Shandong Province Natural Science Foundation (ZR2009CM011). References [1] P.E. Lonning, Study of suboptimum treatment response: lessons from breast cancer, Lancet Oncol. 4 (2003) 177–185. [2] D.J. Bharali, M. Khalil, M. Gurbuz, T.M. Simone, S.A. Mousa, Nanoparticles and cancer therapy: a concise review with emphasis on dendrimers, Int. J. Nanomed. 4 (2009) 1–7. [3] D.B. Fenske, P.R. Cullis, Liposomal nanomedicines, Expert Opin. Drug Deliv. 5 (2008) 25–44. [4] M. Rawat, D. Singh, S. Saraf, S. Saraf, Lipid carrier: a versatile delivery vehicle for proteins and peptides, Yakugaku Zasshi 128 (2008) 269–280. [5] R.H. Müller, M. Radtke, S.A. Wissing, Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Adv. Drug Deliv. Rev. 54 (Suppl. 1) (2002) S131–S155. [6] E.B. Souto, R.H. Müller, Lipid nanoparticles: effect on bioavailability and pharmacokinetic changes, Handb. Exp. Pharmacol. 197 (2010) 115–141. [7] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Control. Release 65 (2000) 271–284. [8] M.C. Bissery, G. Nohynek, G.J. Sanderink, F. Lavelle, Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I. Preclinical experience, Anticancer Drugs 6 (1995) 339–355. [9] E. Saloustros, V. Georgoulias, Docetaxel in the treatment of advanced non-small-cell lung cancer, Expert Rev. Anticancer Ther. 8 (2008) 1207– 1222. [10] E. Saloustros, D. Mavroudis, V. Georgoulias, Paclitaxel and docetaxel in the treatment of breast cancer, Expert Opin. Pharmacother. 9 (2008) 2603– 2616. [11] N.K. Haass, K. Sproesser, T.K. Nguyen, R. Contractor, C.A. Medina, K.L. Nathanson, M. Herlyn, K.S. Smalley, The mitogen-activated protein/extracellular signalregulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel, Clin. Cancer Res. 14 (2008) 230–239. [12] H. Gelderblom, J. Verweij, K. Nooter, A. Sparreboom, Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation, Eur. J. Cancer 37 (2001) 1590–1598.

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