Accepted Manuscript Title: Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions Author: HE Suna YANG Hui ZHANG Ruizhi LI Yan DUAN Lengxin PII: DOI: Reference:
S0378-5173(14)00823-0 http://dx.doi.org/doi:10.1016/j.ijpharm.2014.11.020 IJP 14459
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
30-6-2014 19-10-2014 9-11-2014
Please cite this article as: Suna, HE, Hui, YANG, Ruizhi, ZHANG, Yan, LI, Lengxin, DUAN, Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2014.11.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions HE Suna a,*, YANG Hui a, ZHANG Ruizhi b, LI Yan a, DUAN Lengxin a a Department of Pharmaceutical Sciences, Medical college, Henan University of Science & Technology, Luoyang, 471003, China b Department of Marketing, Henan University of Animal Husbandry and Economy, Zhengzhou, 450045, China
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* Corresponding author: HE Suna, Tel/Fax: +86 379 6482 0862 E-mail address: [email protected] postal address: Department of Pharmaceutical Sciences, Medical college, Henan University of Science & Technology, 31 Anhui Road, Luoyang, Henan, 471003, China.
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Abstract Teniposide (TEN) is a potent, broad spectrum antitumor agent, especially for cerebroma. But the application in clinic was limited because of its poor solubility. In this paper, teniposide nanosuspensions drug delivery system (TEN-NSDDS) for intravenous administration was developed for the first time. Specifically, TEN nanosuspensions were prepared by an anti-solvent sonication-precipitation method and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. TEN nanosuspensions prepared showed rod-like morphology and the size was 151±11 nm with a narrow poly dispersion index 0.138 determined by dynamic light scattering. The obtained TEN nanosuspensions were physically stable at least 10 days at 4 °C. And the freeze-drying preparations were stable during 3 months. The cytotoxicity of TEN nanosuspensions were considerable to that of VUMON against U87MG and C6 cells in vitro. When tested in rats bearing C6 tumors, the TEN concentration in the tumors treated by the nanosuspensions was more than 20 times than that by the TEN solution at 2 h. The TEN nanosuspensions exhibited significant tumor growth inhibition. Overall, the results suggested that nanosuspensions was an alternative formulation for teniposide to be administered intravenously and it would be a promising formulation in clinic. Key words: teniposide; nanosuspensions; anti-solvent sonication-precipitation method; stability; antitumor activity
1. Introduction
Teniposide (TEN) is a semisynthetic derivative of podophyllotoxin resina (McCowage, et al., 1995). Despite numerous studies showing TEN as a promising antitumor compound, its application in clinic was limited because of its poor solubility and unexpected adverse effect (Mane et al., 2004; Attia et al., 2012; Bakheet et al., 2011). To overcome the poor solubility, TEN injection (VUMON) contained solubilizer Cremophor, which caused additional toxic reactions in clinic, such as hypersensitivity (Carstensen et al., 1989; Nolte et al., 1988), hypertension (Shimizu et al.,1987), hypoeosinophilia and hematological
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toxicity (Kubisz et al., 1995). Given the aggrieved solubility problems, several TEN delivery systems have been investigated, including nanoparticles (Zhang et al., 2013; Mo et al., 2012), liposmes (Zhang et al., 2009), submicron lipid particles (Liliemark et al., 1995}, lipid emulsions (Wang et al., 2009) and self-microemulsions (He et al., 2012). However, there were some drawbacks for many of these systems, such as drug leakage, poor physical stability, low drug loading, usage of solubilizing and/or encapsulating excipients which may also lead to adverse side effects. The recent reports showed that nanosuspensions have been widely used to solve problems associated with poor aqueous solubility and low bioavailability (Patel and Agrawal, 2011). The literature reported that Camptothecine (CPT) nanocrystals was stable at least six months and exhibited significant suppression of tumor growth (Zhang et al., 2011). Carvedilol (CAR) nanosuspensions markedly increased the dissolution rate by reducing the size and the pharmacokinetical study demonstrated that the Cmax and AUC0–36 values of CAR nanosuspensions were approximately 3.3- and 2.9-fold greater than that of the commercial CAR tablets, respectively (Liu et al., 2012). Nanosized particles could offer high drug loading, enhance drug solubility, dramaticly increase the dissolution rate of the drug (Dong et al., 2009; Cho et al., 2010), and substantially increase oral bioavailability (Xia et al., 2010). Therefore, nanosuspensions has attracted increased attention in delivering poorly soluble drug, especially for delivering anticancer drugs. Based on all of the above, we tried to develop an TEN nanosuspension drug delivery system(TEN-NSDDS) using the anti-solvent sonication-precipitation method. In the paper, the process parameters and the formulation of TEN nanosuspensions were optimized. The physicochemical characteristics, stability and drug release in vitro were investigated in detail. Finally, the antitumor activity of TEN nanosuspensions in vitro and in vivo were evaluated compared with that of VUMON.
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2 Materials and methods 2.1 Materials
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Teniposide (purity ≥ 98%) was purchased from Hubei prosperity galaxy chemical Co., LTD (Hubei, China). Pluronic F68, Hydroxypropyl Methyl Cellulose (HPMC) and polyvinylpyrrolidone (PVP) K30 were donated by BASF (Shanghai, China). Minimum essential medium (Eagle) with Earls’s BSS (MEM) and fatal bovine serum (FBS) were obtained from Gibco Co. (UK). The antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) and non-essential amino acids (NEAA) were purchased from Sigma-Aldrich Co.(USA). The cell lines U87MG and C6 used in antitumor study was obtained from Chinese Academy of Medical Science(Beijing, China). Methanol of HPLC-grade were obtained from Tedia Company Inc. (Fairfield, OH, USA). All other reagents were of
analytical grade.
2.2 Preparation of TEN nanosuspensions 2.2.1 Preparation method
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TEN nanosusupensions were prepared by anti-solvent sonication-precipitation method. In brief, TEN was completely dissolved in acetone to form organic phase, distilled water containing some polymers as anti-solvent. Then 1 mL of organic solution was quickly injected by syringe into 20 mL of anti-solvent under rapid stirring at 1,000 rpm for 10 min. Immediately, drug particles precipitated when the two phases were mixed. Then the samples were treated with an Ultrasonic Processor (scientz-950E, Ningbo Scientz Biotechnology Co. Ltd., China) to decrease the particle size. The period of ultrasound burst was set to 3 s with a pause of 3 s between two ultrasound bursts. The temperature was controlled at 4-8 °C during the process. The acetone in the samples was removed by rotary evaporation under the vacuum decompression condition and under vacuum at 35 °C for 12 h. For long-term storage of the final product, the freshly prepared TEN nanosuspensions were freeze-dried according to the literature (Liu et al., 2012).
2.2.2. Particle size analysis
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2.2.3. Scanning Electron Microscopy
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The mean particle size (z-average) and polydispersity index (PDI) were determined by dynamic light scattering (DLS) analysis in a Malvern Zetasizer nano ZS90 (Malvern Instruments, UK) at 25 °C. Before the measurement, each sample was vortexed for 5 s to avoid particle settlement and measured in triplicate.
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A scanning electron microscope (SEM) (JSM-6010LA, JEOL, Japan) was used to visualize the morphology of TEN nanosuspensions. Before observation, An SEM sample was fixed onto metal stubs previously secured onto an SEM sample holder. And then the sample was sputter-coated with a conductive layer of gold for approximately 15 nm thick coating under a vacuum.
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2.3. High-performance liquid chromatographic (HPLC) analysis of TEN in vitro
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The content of teniposide in each sample was detected by an HPLC system of LC-20A (Shimadzu, Kyoto, Japan) and an analysis column (octa decyl silane column, 5 µm, 200×4.6 mm). The mobile phase consisted of 45 % acetonitrile and 55 % water. The column was eluted at a flow rate of 1.0 mL/min at 35 °C. The detect wavelength was set at 240 nm. The calibration curve was liner over the range of 0.1 - 5 µg/mL (r=0.9998). The intra-day and inter-day coefficients of variation was less than 2 %. The accuracy of the method was verified with recovery values of 98 % - 102 %.
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2.4. In vitro release To measure release kinetics, the lyophilized preparations of TEN nanosuspensions were dissolved with 5 % glucose to 0.2 mg/mL. 1 mL of the nanosuspensions was put in dialysis bag (molecular weight cut off 6000-8000) that was soaked in double-distilled water for 24 h before use. Then the dialysis bag was immerged in 100 mL of phosphate buffer saline (PBS, pH 7.0 ± 0.1) containing 1.0 % Tween 80 under stirring at 100 rpm (37 ± 0.5 °C). 1 mL of the medium outside the dialysis bag was withdrawn at predetermined intervals of 0.5, 1, 2, 4, 6, 8, 10 h and replaced by 1 mL of fresh release medium. Each
sample was passed through a 0.45 µm filter for HPLC analysis. For comparison, VUMON diluted with 5 % glucose was also tested by the same procedure. All experiments were performed in triplicate.
2.5. Stability study The TEN nanosuspensions were stored at 4 °C. The particle sizes and TEN contents were determined at the time points of 0, 5, 10 and 30 days. The lyophilized preparations of TEN nanosuspensions were stored at 4 °C. At 0, 1, 2, and 3 months, the lyophilized preparations were resuspended with 5 % glucose and then subjected to particle size analysis and drug content determination as described above. The changes in particle size and drug content were recorded. All experiments were performed in triplicate. At 3 months, the lyophilized preparations were resuspended with distilled water for SEM observation.
2.6. Cytotoxicity assay
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The in vitro cytotoxicity of TEN nanosuspensions was tested in gliomas cell lines U87MG and C6 by Sulforhodamine B (SRB) assay, respectively, VUMON as the reference. Cells were cultured in Minimum essential medium (Eagle) with Earls’s BSS (MEM) supplemented with 10 % FBS in a humidified atmosphere containing 5 % CO2 at 37 °C. TEN nanosuspensions and VUMON were diluted with culture medium to the concentration range of 0.05 - 20 µg/mL for measurement. The cells, seeded in 96-well plates at a density of 0.5×104/well, were exposed to the two formulations at 37 °C for 48 h and then subjected to SRB assay. Cells of the control wells were treated with culture medium. The optical densities were measured by a plate reader spectrophotometer (FlexStation 3, Molecular Devices, USA) at 540 nm. Growth inhibition was calculated by following formula.
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Cell Inhibition(%) =
ODc - OD t × 100% ODc - OD b
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Here, ODc was the mean absorption value of the controls, ODt was the mean absorption value of the wells treated with TEN nanosuspensions or VUMON, and ODb was that of the blank wells.
2.7. In vivo antitumor measurement
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Wistar rats, weighting 190±10 g, were obtained from Experimental Animal Center of Henan University of Science &Technology (Henan, China). All the animals were pathogen free and allowed to access food and water freely. All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care of Henan University of Science & Technology. 0.2 mL of C6 cells (5×106/mL) was inoculated subcutaneously to each mouse at the right groin to obtain glioma models. The C6 bearing rats were randomly grouped (6 for control, 6 for VUMON, and 6 for TEN nanosuspensions). 5 % glucose was used as negative control. The dose was 10 mg/Kg for VUMON or TEN nanosuspensions. When the tumor volume reached approximately 100 mm3, a respective treatment was given to each animal via tail vein for three times, once every three days. Tumor sizes were monitored daily with a caliper in two dimensions (length and width). The tumor volume was estimated as length×(width)2/2. The rats were euthanized on day 15 after the first administration and tumors were excised and weighed. And then the photos of the excised tumors were taken. Tumor inhibition rate (IR) was
calculated as:
IR(%) =
Wc - Wt × 100% Wc
Here, W c was the average tumor weight of rats adminstrated 5 % glucose and Wt was the average tumor weight of rats treated by VUMON or TEN nanosuspensions, respectively. To determine the biodistribution of TEN in tumors, the above rats bearing tumors were also used. The TEN nanosuspensions or VUMON were intravenously administered to the rats (6 per group) via tail veins, respectively, at a dose of 10 mg/Kg when the tumor volume reached approximately 500 mm3. Blood collection, organs and tumor samples treatment, and drug analysis in each sample were the same as the earlier reported (He et al., 2012).
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2.8. Statistical analysis
3. Results and discussion 3.1. TEN nanosuspensions preparation
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Quantitative data were expressed as mean ± standard deviation (SD). Statistical significance between treatment and control group was evaluated by Student's t test. A p value less than 0.05, the difference was considered to be significant, while less than 0.01 was highly significant.
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To facilitate the removal of the organic solvent, acetone was selected as the solvent although teniposide dissolves much more in DMSO or DMA than acetone. Additionally, pH 6.0 - 6.5 water was specifically selected as the anti-solvent system and the storage medium due to the fact that it is the most stable pH range for teniposide (Wang et al. 2009). Long-chain polymeric surfactants which were used as the stabilizers in nanosuspension drug delivery system could reduce Ostwald’s ripening and increase the physical stability of nanocrystals(Wang et al., 2013; Sudhakar et al., 2014). Various stabilizers introduced to the system, the shape and size of the nanocrystals could be altered (Zhang et al., 2011). In the study, several polymers were tested and PVP was shown to have a capability of stabilizing the size of TEN nanosuspensions (data not shown). The effect of TEN concentration in organic phase and the PVP concentration in water pahse on the size of TEN nanosuspensions were shown in Fig.1a and Fig.1b, when TEN concentration changed from 5 to 20 mg/ml, the size of TEN nanosuspensions decreased at the first and then increased, which was induced mainly by the drug supersaturation. In order to form crystals of similar size, high supersaturation was needed to form nucleation swiftly (Kakran et al., 2010). However, a higher drug level could accelerate crystal growth by promoting agglomeration and/or coagulation although the stabilizer was involved in the formulation (Xia et al., 2010). As such, crystals with expected size could be achieved by choosing a proper drug concentration, besides the combination of good and anti-solvents. In Fig. 1b, with time extending, the particle size changed slightly with the PVP concentration ranging from 0. 05 % - 0.15 %. But the size increased with PVP concentration increment. PVP maybe remained in the interfacial area between the drug particles and the surrounding solution, thereby preventing the nanosuspensions from
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adhering to each other (Van Eerdenbrugh et al., 2008). When the concentration of PVP was low, it was not enough to fully cover the newly formed surfaces of the nanosuspensions or effectively inhibit the Ostwald ripening process, resulting in the growth of particle size. However, an excessive amount of PVP could increase the particle size by thickening the coat and inhibit the diffusion between the solvent and the anti-solvent during precipitation. Therefore, PVP concentration 0.1 % was selected in the study. Sonication was utilized to produce uniform, nanosized crystals within a narrow size distribution (Kaerger and Price, 2004). Sonication was capable of creating a large number of nuclei simultaneously, crucial for producing uniform nanosuspensions, and then decreasing the size (Guo et al., 2005). From Fig.1c we could see that with the power increased, the size of the TEN nanosuspensions showed an initial decrease and then a negligible change. The phenomenon was also appeared in Fig.1d. That is to say, the power and the time of treatment exceeded 300 W and 3 min respectively were needless to decrease the nanosuspensions size. By the single factor experiments, the formulation and process conditions of TEN nanosuspensions were optimized. Briefly, 1mL of 10 mg/mL TEN acetone solution was quickly injected into 20 mL of 0.1 % PVP water solution under rapid stirring at 1,000 rpm for 10 min. Then the samples were treated with ultrasonication at 300 W for 3 min. Finally, the acetone in the samples was removed by rotary evaporation under the vacuum decompression condition and under vacuum at 35 °C for 12 h. TEN nanosuspensions prepared with the optimized formulation showed rod-like morphology and the size ranged from 200 to 500 nm as shown in Fig. 2. The size determined by DLS was 151 ± 11 nm with a narrow PDI 0.138. Note that the particle size determined by DLS is more accurate for spherical particles. For high aspect-ratio particles, the size measured by DLS was inconsistent with that observed under SEM. However, the method may still offer useful information during formulation optimization (Panchal et al., 2014).
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3.2. In vitro release of TEN nanosuspensions
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Buffer medium with 1.0 % Tween 80 was used to ensure sink condition during the release test. Fig.3 represents a comparison of the release profiles of VUMON and TEN nanosuspensions. The results revealed that passage of TEN molecule in the nanosuspensions through the dialysis membrane was considerably slower as compared with that of VUMON. VUMON released up to 94.01% within 4 hours. In contrast, 96.41% of the TEN nanosuspensions released from the dialysis bags needed 8 hours. The slow release rate of TEN nanosuspensions could be attributed to the slowly soluiton of teniposide. Teniposide dissolved in VUMON. While, it existed in nanosuspensions in the form of nanocrysals. During release, teniposide nanosuspensions needed to dissolve at first and then pass through the dialysis bags. However, the slower release rate maybe adds to the benefit of prolonging the system circulation of TEN for chemotherapy. Because in vivo TEN nanosuspensions could permeate into the tumors and get trapped due to the enhanced permeability and retention (EPR) effect, and then slowly release into the system circulation (Maruyama, 2011).
3.3. Storage stability The short-term stability of TEN nanosuspensions was investigated at 4 °C. At day 0, 5,
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10 and 30, the mean particle size and drug content was measured. As shown in table 1, the changes of the particle sizes and drug contents were negligible during 10 days. However, at 30 days, the TEN nanosuspensions appeared slight precipitation and the teniposide content decreased. That is to say, the TEN nanosuspensions was unsuitable for long-term storage. But it was stable enough to experienced freeze-drying processing during 10 days. In order to enhance the stability of TEN nanosuspensions, freeze-drying technology was utilized, and the stability of the freeze-drying preparations of TEN nanosuspensions were studied at 4 °C. At the pre-determined time points, the freeze-drying powder of TEN nanosuspensions was resuspended. Particle size, PDI, and TEN content monitored showed no significant change over the period of three months (table 1). The morphology of TEN nanosuspensions lyophilized preparation and the sample at three months were observed by SEM. From the SEM, we could see that TEN nanosuspensions were rod-like and the morphology have no change after freeze-drying and during the stability study. In the stability study, the slight precipitation of TEN nanosuspensions could be explained by the Ostwald ripening phenomenon. Small particles in the suspensions would dissolve and molecules are re-deposited to larger particles. And then the precipitation would appear. From another point of view, teniposide in aqueous solution would degrade. The previous literature reported that the degradation of teniposide in aqueous solution was shown to follow pseudo-first-order degradation kinetics (Wang et al., 2009). The half-life (t(1/2)) of teniposide in aqueous solution was 2.6 days at 10 °C under the most stable pH range of 6.0 - 6.5. Although at a lower temperature, the degradation would postpone, the degradation of teniposide in the nanosuspensions was observed in our experience. The physical and chemical stability of teniposide nanosuspensions were guaranteed by freeze-drying technology. 3.4. Cytotoxicity of TEN nanosuspensions Cytotoxicity of TEN nanosuspensions was evaluated in cancerous U87MG and C6 cell lines, and compared with that of VUMON. As shown in Fig. 4, the cytotoxic activity of TEN nanosuspensions was dose-dependent both for U87MG and C6 cells. The half maximal inhibitory concentrations (IC50) were determined from the cell viability curves. Of TEN nanosuspensions, IC50 were 1.53±0.60 µg/mL and 0.481±0.033 µg/mL against U87MG and C6 cells, respectively. The corresponding values of VUMON were 1.473±1.819 µg/mL and 0.386±0.024 µg/mL. There was no statistically significant difference between TEN nanosuspensions and VUMON both in C6 and U87MG cells (p > 0.05). However, the IC50 of TEN nanosuspension were higher than that of VUMON both in U87MG and C6 cells. Additionally, it seems that C6 cells was more sensitive to teniposide. The results could be explained from two aspects. On the one hand, in the cytotoxicity experiments, the formulations were incubated with cells for 48 h before the results were analyzed. Therefore, a majority of TEN nanosuspensions were likely dissolved in the medium, which resulted in a similar cytotoxic performance with that of teniposide solution. On the other hand, the solvents in VUMON, especially Cremophor were toxic to some extent. Cremophor maybe induce the cell necrosis through some approach (Iwase et al., 2004; Slater et al., 1995; Yamaguchi et al., 2005).
3.5. In vivo antitumor effect of TEN nanosuspensions
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The antitumor activity of TEN nanosuspensions were evaluated in Wistar rats bearing C6 gliomas tumors. The results were shown in Fig. 5. From the tumor size chart (Fig. 5a), we could see that along with time extending, both groups treated by TEN nanosuspensions and VUMON yielded highly significant tumor inhibition compared with the 5 % glucose group (p ˂ 0.01). What’s more, compared to VUMON group, the tumor progression in the group treated by TEN nanosuspensions was especially slower (p ˂ 0.05). Additionally, from day 0 to day 7, the average tumor size in TEN nanosuspensions was considerable to that in VUMON group. While, the average tumor size in TEN nanosuspensions was smaller than that in VUMON group from day 8 to 13. Tumor inhibition rates were calculated on the basis of the averaged weight of the tumors, which were obtained by dissecting the rats bearing C6 tumors on day 15 after the first administration. The results shown in Fig. 5b further indicated the better antitumor effect by TEN nanosuspensions. The average tumor weights was 1.683, 0.618 and 0.721 g respectively for groups treated with 5 % glucose, TEN nanosuspensions and VUMON, respectively. The inhibition rates were 69.2% and 57.2% for TEN nanosuspensions and VUMON relative to 5 % glucose group, respectively, which were significantly different (p < 0.05). And the photos of the excised tumors visually reflects the difference between the three groups (Fig. 5c). The above antitumor measurement proved that TEN nanosuspensions had significantly greater antitumor activity than VUMON. This could be mainly attributed to the EPR effect (Hobbs et al., 1998). Upon tail vein injection, drug solution were directly in contact with blood and cleared from the system circle fast. In contrast, TEN nanosuspensions were prone to permeate into the tumor and get trapped. Then, drug molecules in the nanosuspensions slowly released to achieve prolonged circulation in the plasma, which was consistent with the results of release in vitro. From this respect, TEN nanosuspensions had greater advantage than teniposide solution.
3.6. TEN biodistribution in tumor-bearing rats
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Biodistribution of TEN in the tumor, plasma, and major organs were examined after the treatment by VUMON or TEN nanosuspensions at a comparable dose of 10 mg/Kg. Shown in Fig. 6, at 0.5 h after the injection, the concentrations of TEN in the plasma, tumor and other tissues, except lung, were higher than or similar to its control. But, in the lung, TEN was highly accumulated after the nanosuspensions treatment, which could be caused by some relatively large particles. The phenomenon seems against the material balance law. The average TEN concentration in VUMON were lower than that in TEN nanosuspensions in plasma, liver and kidney at 0.5h. But the standard deviation (SD) in VUMON group was higher than that in TEN nanosuspensions group, which could be seen from Fig.6. Although the method for teniposide determination in biological samples had been validated. Therefore, the phenomenon could be explained by the higher SD. The tissue distribution was keeping with the material balance law in each rat. At 2 h, except in the plasma, the TEN concentrations in the tumor and tissues were higher treated by the TEN nanosuspensions than that by VUMON. More interestingly, the TEN accumulation in the tumor treated by the nanosuspensions was markedly higher, by more than 20 folds, than that by the solution. The phenomenon could be attributed to the larger clearance of VUMON, EPR effect and sustained release of TEN nanosuspensions. Generally, the
clearance of solution in vivo was larger than the nanocarrier drug delivery system (He et al., 2012), which lead to the less total drug in VUMON group at 2h. TEN nanosuspensions, as a passive targeted drug delivery system, permeated into tumors and got trapped due to EPR effect, and distributed into the tissues like liver, spleen, and lung, where phagocytosis cells are rich (Stylianopoulos, 2013). The larger accumulation in the tumor (Fig. 6) could be used to explain the better antitumor effect by using the drug nanosuspensions in the antitumor effect study. Orthotopic brain tumor model was better to evaluate TEN nanosuspensions. Unfortunately, due to technical reasons, orthotopic brain tumor model was not established and the drug concentrations in the tumor and other major organs were not detected at the longer time points, such as 4 h, 8 h.
4. Conclusions
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In this study, a novel teniposide formulation for intravenous administration, TEN nanosuspensions, was proposed and successfully prepared via the anti-solvent precipitation-sonication technique, PVP as the stabilizer. With the optimized formulation, TEN nanosuspensions with a mean particle size of 151 ± 11 nm and a narrow PDI 0.138 were obtained. And TEN nanosuspensions were rod-like under SEM. The obtained TEN nanosuspensions was physically and chemically stable at least 10 days at 4°C. And the lyophilized preparations were stable during 3 months. In vitro cytotoxicity study showed that TEN nanosuspensions could induce similar, if not better, cell growth inhibition for U87MG and C6, compared with VUMON. While, the in vivo antitumor test in C6 tumors bearing rats demonstrated that the inhibition rates were 69.2% and 57.2% for TEN nanosuspensions and VUMON relative to 5 % glucose group, respectively. The antitumor effect of TEN nanosuspensions was markedly superior to VUMON (p
S0378-5173(14)00823-0 http://dx.doi.org/doi:10.1016/j.ijpharm.2014.11.020 IJP 14459
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
30-6-2014 19-10-2014 9-11-2014
Please cite this article as: Suna, HE, Hui, YANG, Ruizhi, ZHANG, Yan, LI, Lengxin, DUAN, Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2014.11.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions HE Suna a,*, YANG Hui a, ZHANG Ruizhi b, LI Yan a, DUAN Lengxin a a Department of Pharmaceutical Sciences, Medical college, Henan University of Science & Technology, Luoyang, 471003, China b Department of Marketing, Henan University of Animal Husbandry and Economy, Zhengzhou, 450045, China
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* Corresponding author: HE Suna, Tel/Fax: +86 379 6482 0862 E-mail address: [email protected] postal address: Department of Pharmaceutical Sciences, Medical college, Henan University of Science & Technology, 31 Anhui Road, Luoyang, Henan, 471003, China.
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Abstract Teniposide (TEN) is a potent, broad spectrum antitumor agent, especially for cerebroma. But the application in clinic was limited because of its poor solubility. In this paper, teniposide nanosuspensions drug delivery system (TEN-NSDDS) for intravenous administration was developed for the first time. Specifically, TEN nanosuspensions were prepared by an anti-solvent sonication-precipitation method and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. TEN nanosuspensions prepared showed rod-like morphology and the size was 151±11 nm with a narrow poly dispersion index 0.138 determined by dynamic light scattering. The obtained TEN nanosuspensions were physically stable at least 10 days at 4 °C. And the freeze-drying preparations were stable during 3 months. The cytotoxicity of TEN nanosuspensions were considerable to that of VUMON against U87MG and C6 cells in vitro. When tested in rats bearing C6 tumors, the TEN concentration in the tumors treated by the nanosuspensions was more than 20 times than that by the TEN solution at 2 h. The TEN nanosuspensions exhibited significant tumor growth inhibition. Overall, the results suggested that nanosuspensions was an alternative formulation for teniposide to be administered intravenously and it would be a promising formulation in clinic. Key words: teniposide; nanosuspensions; anti-solvent sonication-precipitation method; stability; antitumor activity
1. Introduction
Teniposide (TEN) is a semisynthetic derivative of podophyllotoxin resina (McCowage, et al., 1995). Despite numerous studies showing TEN as a promising antitumor compound, its application in clinic was limited because of its poor solubility and unexpected adverse effect (Mane et al., 2004; Attia et al., 2012; Bakheet et al., 2011). To overcome the poor solubility, TEN injection (VUMON) contained solubilizer Cremophor, which caused additional toxic reactions in clinic, such as hypersensitivity (Carstensen et al., 1989; Nolte et al., 1988), hypertension (Shimizu et al.,1987), hypoeosinophilia and hematological
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toxicity (Kubisz et al., 1995). Given the aggrieved solubility problems, several TEN delivery systems have been investigated, including nanoparticles (Zhang et al., 2013; Mo et al., 2012), liposmes (Zhang et al., 2009), submicron lipid particles (Liliemark et al., 1995}, lipid emulsions (Wang et al., 2009) and self-microemulsions (He et al., 2012). However, there were some drawbacks for many of these systems, such as drug leakage, poor physical stability, low drug loading, usage of solubilizing and/or encapsulating excipients which may also lead to adverse side effects. The recent reports showed that nanosuspensions have been widely used to solve problems associated with poor aqueous solubility and low bioavailability (Patel and Agrawal, 2011). The literature reported that Camptothecine (CPT) nanocrystals was stable at least six months and exhibited significant suppression of tumor growth (Zhang et al., 2011). Carvedilol (CAR) nanosuspensions markedly increased the dissolution rate by reducing the size and the pharmacokinetical study demonstrated that the Cmax and AUC0–36 values of CAR nanosuspensions were approximately 3.3- and 2.9-fold greater than that of the commercial CAR tablets, respectively (Liu et al., 2012). Nanosized particles could offer high drug loading, enhance drug solubility, dramaticly increase the dissolution rate of the drug (Dong et al., 2009; Cho et al., 2010), and substantially increase oral bioavailability (Xia et al., 2010). Therefore, nanosuspensions has attracted increased attention in delivering poorly soluble drug, especially for delivering anticancer drugs. Based on all of the above, we tried to develop an TEN nanosuspension drug delivery system(TEN-NSDDS) using the anti-solvent sonication-precipitation method. In the paper, the process parameters and the formulation of TEN nanosuspensions were optimized. The physicochemical characteristics, stability and drug release in vitro were investigated in detail. Finally, the antitumor activity of TEN nanosuspensions in vitro and in vivo were evaluated compared with that of VUMON.
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2 Materials and methods 2.1 Materials
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Teniposide (purity ≥ 98%) was purchased from Hubei prosperity galaxy chemical Co., LTD (Hubei, China). Pluronic F68, Hydroxypropyl Methyl Cellulose (HPMC) and polyvinylpyrrolidone (PVP) K30 were donated by BASF (Shanghai, China). Minimum essential medium (Eagle) with Earls’s BSS (MEM) and fatal bovine serum (FBS) were obtained from Gibco Co. (UK). The antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) and non-essential amino acids (NEAA) were purchased from Sigma-Aldrich Co.(USA). The cell lines U87MG and C6 used in antitumor study was obtained from Chinese Academy of Medical Science(Beijing, China). Methanol of HPLC-grade were obtained from Tedia Company Inc. (Fairfield, OH, USA). All other reagents were of
analytical grade.
2.2 Preparation of TEN nanosuspensions 2.2.1 Preparation method
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TEN nanosusupensions were prepared by anti-solvent sonication-precipitation method. In brief, TEN was completely dissolved in acetone to form organic phase, distilled water containing some polymers as anti-solvent. Then 1 mL of organic solution was quickly injected by syringe into 20 mL of anti-solvent under rapid stirring at 1,000 rpm for 10 min. Immediately, drug particles precipitated when the two phases were mixed. Then the samples were treated with an Ultrasonic Processor (scientz-950E, Ningbo Scientz Biotechnology Co. Ltd., China) to decrease the particle size. The period of ultrasound burst was set to 3 s with a pause of 3 s between two ultrasound bursts. The temperature was controlled at 4-8 °C during the process. The acetone in the samples was removed by rotary evaporation under the vacuum decompression condition and under vacuum at 35 °C for 12 h. For long-term storage of the final product, the freshly prepared TEN nanosuspensions were freeze-dried according to the literature (Liu et al., 2012).
2.2.2. Particle size analysis
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2.2.3. Scanning Electron Microscopy
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The mean particle size (z-average) and polydispersity index (PDI) were determined by dynamic light scattering (DLS) analysis in a Malvern Zetasizer nano ZS90 (Malvern Instruments, UK) at 25 °C. Before the measurement, each sample was vortexed for 5 s to avoid particle settlement and measured in triplicate.
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A scanning electron microscope (SEM) (JSM-6010LA, JEOL, Japan) was used to visualize the morphology of TEN nanosuspensions. Before observation, An SEM sample was fixed onto metal stubs previously secured onto an SEM sample holder. And then the sample was sputter-coated with a conductive layer of gold for approximately 15 nm thick coating under a vacuum.
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2.3. High-performance liquid chromatographic (HPLC) analysis of TEN in vitro
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The content of teniposide in each sample was detected by an HPLC system of LC-20A (Shimadzu, Kyoto, Japan) and an analysis column (octa decyl silane column, 5 µm, 200×4.6 mm). The mobile phase consisted of 45 % acetonitrile and 55 % water. The column was eluted at a flow rate of 1.0 mL/min at 35 °C. The detect wavelength was set at 240 nm. The calibration curve was liner over the range of 0.1 - 5 µg/mL (r=0.9998). The intra-day and inter-day coefficients of variation was less than 2 %. The accuracy of the method was verified with recovery values of 98 % - 102 %.
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2.4. In vitro release To measure release kinetics, the lyophilized preparations of TEN nanosuspensions were dissolved with 5 % glucose to 0.2 mg/mL. 1 mL of the nanosuspensions was put in dialysis bag (molecular weight cut off 6000-8000) that was soaked in double-distilled water for 24 h before use. Then the dialysis bag was immerged in 100 mL of phosphate buffer saline (PBS, pH 7.0 ± 0.1) containing 1.0 % Tween 80 under stirring at 100 rpm (37 ± 0.5 °C). 1 mL of the medium outside the dialysis bag was withdrawn at predetermined intervals of 0.5, 1, 2, 4, 6, 8, 10 h and replaced by 1 mL of fresh release medium. Each
sample was passed through a 0.45 µm filter for HPLC analysis. For comparison, VUMON diluted with 5 % glucose was also tested by the same procedure. All experiments were performed in triplicate.
2.5. Stability study The TEN nanosuspensions were stored at 4 °C. The particle sizes and TEN contents were determined at the time points of 0, 5, 10 and 30 days. The lyophilized preparations of TEN nanosuspensions were stored at 4 °C. At 0, 1, 2, and 3 months, the lyophilized preparations were resuspended with 5 % glucose and then subjected to particle size analysis and drug content determination as described above. The changes in particle size and drug content were recorded. All experiments were performed in triplicate. At 3 months, the lyophilized preparations were resuspended with distilled water for SEM observation.
2.6. Cytotoxicity assay
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The in vitro cytotoxicity of TEN nanosuspensions was tested in gliomas cell lines U87MG and C6 by Sulforhodamine B (SRB) assay, respectively, VUMON as the reference. Cells were cultured in Minimum essential medium (Eagle) with Earls’s BSS (MEM) supplemented with 10 % FBS in a humidified atmosphere containing 5 % CO2 at 37 °C. TEN nanosuspensions and VUMON were diluted with culture medium to the concentration range of 0.05 - 20 µg/mL for measurement. The cells, seeded in 96-well plates at a density of 0.5×104/well, were exposed to the two formulations at 37 °C for 48 h and then subjected to SRB assay. Cells of the control wells were treated with culture medium. The optical densities were measured by a plate reader spectrophotometer (FlexStation 3, Molecular Devices, USA) at 540 nm. Growth inhibition was calculated by following formula.
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Cell Inhibition(%) =
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Here, ODc was the mean absorption value of the controls, ODt was the mean absorption value of the wells treated with TEN nanosuspensions or VUMON, and ODb was that of the blank wells.
2.7. In vivo antitumor measurement
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Wistar rats, weighting 190±10 g, were obtained from Experimental Animal Center of Henan University of Science &Technology (Henan, China). All the animals were pathogen free and allowed to access food and water freely. All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care of Henan University of Science & Technology. 0.2 mL of C6 cells (5×106/mL) was inoculated subcutaneously to each mouse at the right groin to obtain glioma models. The C6 bearing rats were randomly grouped (6 for control, 6 for VUMON, and 6 for TEN nanosuspensions). 5 % glucose was used as negative control. The dose was 10 mg/Kg for VUMON or TEN nanosuspensions. When the tumor volume reached approximately 100 mm3, a respective treatment was given to each animal via tail vein for three times, once every three days. Tumor sizes were monitored daily with a caliper in two dimensions (length and width). The tumor volume was estimated as length×(width)2/2. The rats were euthanized on day 15 after the first administration and tumors were excised and weighed. And then the photos of the excised tumors were taken. Tumor inhibition rate (IR) was
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IR(%) =
Wc - Wt × 100% Wc
Here, W c was the average tumor weight of rats adminstrated 5 % glucose and Wt was the average tumor weight of rats treated by VUMON or TEN nanosuspensions, respectively. To determine the biodistribution of TEN in tumors, the above rats bearing tumors were also used. The TEN nanosuspensions or VUMON were intravenously administered to the rats (6 per group) via tail veins, respectively, at a dose of 10 mg/Kg when the tumor volume reached approximately 500 mm3. Blood collection, organs and tumor samples treatment, and drug analysis in each sample were the same as the earlier reported (He et al., 2012).
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2.8. Statistical analysis
3. Results and discussion 3.1. TEN nanosuspensions preparation
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Quantitative data were expressed as mean ± standard deviation (SD). Statistical significance between treatment and control group was evaluated by Student's t test. A p value less than 0.05, the difference was considered to be significant, while less than 0.01 was highly significant.
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To facilitate the removal of the organic solvent, acetone was selected as the solvent although teniposide dissolves much more in DMSO or DMA than acetone. Additionally, pH 6.0 - 6.5 water was specifically selected as the anti-solvent system and the storage medium due to the fact that it is the most stable pH range for teniposide (Wang et al. 2009). Long-chain polymeric surfactants which were used as the stabilizers in nanosuspension drug delivery system could reduce Ostwald’s ripening and increase the physical stability of nanocrystals(Wang et al., 2013; Sudhakar et al., 2014). Various stabilizers introduced to the system, the shape and size of the nanocrystals could be altered (Zhang et al., 2011). In the study, several polymers were tested and PVP was shown to have a capability of stabilizing the size of TEN nanosuspensions (data not shown). The effect of TEN concentration in organic phase and the PVP concentration in water pahse on the size of TEN nanosuspensions were shown in Fig.1a and Fig.1b, when TEN concentration changed from 5 to 20 mg/ml, the size of TEN nanosuspensions decreased at the first and then increased, which was induced mainly by the drug supersaturation. In order to form crystals of similar size, high supersaturation was needed to form nucleation swiftly (Kakran et al., 2010). However, a higher drug level could accelerate crystal growth by promoting agglomeration and/or coagulation although the stabilizer was involved in the formulation (Xia et al., 2010). As such, crystals with expected size could be achieved by choosing a proper drug concentration, besides the combination of good and anti-solvents. In Fig. 1b, with time extending, the particle size changed slightly with the PVP concentration ranging from 0. 05 % - 0.15 %. But the size increased with PVP concentration increment. PVP maybe remained in the interfacial area between the drug particles and the surrounding solution, thereby preventing the nanosuspensions from
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adhering to each other (Van Eerdenbrugh et al., 2008). When the concentration of PVP was low, it was not enough to fully cover the newly formed surfaces of the nanosuspensions or effectively inhibit the Ostwald ripening process, resulting in the growth of particle size. However, an excessive amount of PVP could increase the particle size by thickening the coat and inhibit the diffusion between the solvent and the anti-solvent during precipitation. Therefore, PVP concentration 0.1 % was selected in the study. Sonication was utilized to produce uniform, nanosized crystals within a narrow size distribution (Kaerger and Price, 2004). Sonication was capable of creating a large number of nuclei simultaneously, crucial for producing uniform nanosuspensions, and then decreasing the size (Guo et al., 2005). From Fig.1c we could see that with the power increased, the size of the TEN nanosuspensions showed an initial decrease and then a negligible change. The phenomenon was also appeared in Fig.1d. That is to say, the power and the time of treatment exceeded 300 W and 3 min respectively were needless to decrease the nanosuspensions size. By the single factor experiments, the formulation and process conditions of TEN nanosuspensions were optimized. Briefly, 1mL of 10 mg/mL TEN acetone solution was quickly injected into 20 mL of 0.1 % PVP water solution under rapid stirring at 1,000 rpm for 10 min. Then the samples were treated with ultrasonication at 300 W for 3 min. Finally, the acetone in the samples was removed by rotary evaporation under the vacuum decompression condition and under vacuum at 35 °C for 12 h. TEN nanosuspensions prepared with the optimized formulation showed rod-like morphology and the size ranged from 200 to 500 nm as shown in Fig. 2. The size determined by DLS was 151 ± 11 nm with a narrow PDI 0.138. Note that the particle size determined by DLS is more accurate for spherical particles. For high aspect-ratio particles, the size measured by DLS was inconsistent with that observed under SEM. However, the method may still offer useful information during formulation optimization (Panchal et al., 2014).
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3.2. In vitro release of TEN nanosuspensions
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Buffer medium with 1.0 % Tween 80 was used to ensure sink condition during the release test. Fig.3 represents a comparison of the release profiles of VUMON and TEN nanosuspensions. The results revealed that passage of TEN molecule in the nanosuspensions through the dialysis membrane was considerably slower as compared with that of VUMON. VUMON released up to 94.01% within 4 hours. In contrast, 96.41% of the TEN nanosuspensions released from the dialysis bags needed 8 hours. The slow release rate of TEN nanosuspensions could be attributed to the slowly soluiton of teniposide. Teniposide dissolved in VUMON. While, it existed in nanosuspensions in the form of nanocrysals. During release, teniposide nanosuspensions needed to dissolve at first and then pass through the dialysis bags. However, the slower release rate maybe adds to the benefit of prolonging the system circulation of TEN for chemotherapy. Because in vivo TEN nanosuspensions could permeate into the tumors and get trapped due to the enhanced permeability and retention (EPR) effect, and then slowly release into the system circulation (Maruyama, 2011).
3.3. Storage stability The short-term stability of TEN nanosuspensions was investigated at 4 °C. At day 0, 5,
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10 and 30, the mean particle size and drug content was measured. As shown in table 1, the changes of the particle sizes and drug contents were negligible during 10 days. However, at 30 days, the TEN nanosuspensions appeared slight precipitation and the teniposide content decreased. That is to say, the TEN nanosuspensions was unsuitable for long-term storage. But it was stable enough to experienced freeze-drying processing during 10 days. In order to enhance the stability of TEN nanosuspensions, freeze-drying technology was utilized, and the stability of the freeze-drying preparations of TEN nanosuspensions were studied at 4 °C. At the pre-determined time points, the freeze-drying powder of TEN nanosuspensions was resuspended. Particle size, PDI, and TEN content monitored showed no significant change over the period of three months (table 1). The morphology of TEN nanosuspensions lyophilized preparation and the sample at three months were observed by SEM. From the SEM, we could see that TEN nanosuspensions were rod-like and the morphology have no change after freeze-drying and during the stability study. In the stability study, the slight precipitation of TEN nanosuspensions could be explained by the Ostwald ripening phenomenon. Small particles in the suspensions would dissolve and molecules are re-deposited to larger particles. And then the precipitation would appear. From another point of view, teniposide in aqueous solution would degrade. The previous literature reported that the degradation of teniposide in aqueous solution was shown to follow pseudo-first-order degradation kinetics (Wang et al., 2009). The half-life (t(1/2)) of teniposide in aqueous solution was 2.6 days at 10 °C under the most stable pH range of 6.0 - 6.5. Although at a lower temperature, the degradation would postpone, the degradation of teniposide in the nanosuspensions was observed in our experience. The physical and chemical stability of teniposide nanosuspensions were guaranteed by freeze-drying technology. 3.4. Cytotoxicity of TEN nanosuspensions Cytotoxicity of TEN nanosuspensions was evaluated in cancerous U87MG and C6 cell lines, and compared with that of VUMON. As shown in Fig. 4, the cytotoxic activity of TEN nanosuspensions was dose-dependent both for U87MG and C6 cells. The half maximal inhibitory concentrations (IC50) were determined from the cell viability curves. Of TEN nanosuspensions, IC50 were 1.53±0.60 µg/mL and 0.481±0.033 µg/mL against U87MG and C6 cells, respectively. The corresponding values of VUMON were 1.473±1.819 µg/mL and 0.386±0.024 µg/mL. There was no statistically significant difference between TEN nanosuspensions and VUMON both in C6 and U87MG cells (p > 0.05). However, the IC50 of TEN nanosuspension were higher than that of VUMON both in U87MG and C6 cells. Additionally, it seems that C6 cells was more sensitive to teniposide. The results could be explained from two aspects. On the one hand, in the cytotoxicity experiments, the formulations were incubated with cells for 48 h before the results were analyzed. Therefore, a majority of TEN nanosuspensions were likely dissolved in the medium, which resulted in a similar cytotoxic performance with that of teniposide solution. On the other hand, the solvents in VUMON, especially Cremophor were toxic to some extent. Cremophor maybe induce the cell necrosis through some approach (Iwase et al., 2004; Slater et al., 1995; Yamaguchi et al., 2005).
3.5. In vivo antitumor effect of TEN nanosuspensions
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The antitumor activity of TEN nanosuspensions were evaluated in Wistar rats bearing C6 gliomas tumors. The results were shown in Fig. 5. From the tumor size chart (Fig. 5a), we could see that along with time extending, both groups treated by TEN nanosuspensions and VUMON yielded highly significant tumor inhibition compared with the 5 % glucose group (p ˂ 0.01). What’s more, compared to VUMON group, the tumor progression in the group treated by TEN nanosuspensions was especially slower (p ˂ 0.05). Additionally, from day 0 to day 7, the average tumor size in TEN nanosuspensions was considerable to that in VUMON group. While, the average tumor size in TEN nanosuspensions was smaller than that in VUMON group from day 8 to 13. Tumor inhibition rates were calculated on the basis of the averaged weight of the tumors, which were obtained by dissecting the rats bearing C6 tumors on day 15 after the first administration. The results shown in Fig. 5b further indicated the better antitumor effect by TEN nanosuspensions. The average tumor weights was 1.683, 0.618 and 0.721 g respectively for groups treated with 5 % glucose, TEN nanosuspensions and VUMON, respectively. The inhibition rates were 69.2% and 57.2% for TEN nanosuspensions and VUMON relative to 5 % glucose group, respectively, which were significantly different (p < 0.05). And the photos of the excised tumors visually reflects the difference between the three groups (Fig. 5c). The above antitumor measurement proved that TEN nanosuspensions had significantly greater antitumor activity than VUMON. This could be mainly attributed to the EPR effect (Hobbs et al., 1998). Upon tail vein injection, drug solution were directly in contact with blood and cleared from the system circle fast. In contrast, TEN nanosuspensions were prone to permeate into the tumor and get trapped. Then, drug molecules in the nanosuspensions slowly released to achieve prolonged circulation in the plasma, which was consistent with the results of release in vitro. From this respect, TEN nanosuspensions had greater advantage than teniposide solution.
3.6. TEN biodistribution in tumor-bearing rats
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Biodistribution of TEN in the tumor, plasma, and major organs were examined after the treatment by VUMON or TEN nanosuspensions at a comparable dose of 10 mg/Kg. Shown in Fig. 6, at 0.5 h after the injection, the concentrations of TEN in the plasma, tumor and other tissues, except lung, were higher than or similar to its control. But, in the lung, TEN was highly accumulated after the nanosuspensions treatment, which could be caused by some relatively large particles. The phenomenon seems against the material balance law. The average TEN concentration in VUMON were lower than that in TEN nanosuspensions in plasma, liver and kidney at 0.5h. But the standard deviation (SD) in VUMON group was higher than that in TEN nanosuspensions group, which could be seen from Fig.6. Although the method for teniposide determination in biological samples had been validated. Therefore, the phenomenon could be explained by the higher SD. The tissue distribution was keeping with the material balance law in each rat. At 2 h, except in the plasma, the TEN concentrations in the tumor and tissues were higher treated by the TEN nanosuspensions than that by VUMON. More interestingly, the TEN accumulation in the tumor treated by the nanosuspensions was markedly higher, by more than 20 folds, than that by the solution. The phenomenon could be attributed to the larger clearance of VUMON, EPR effect and sustained release of TEN nanosuspensions. Generally, the
clearance of solution in vivo was larger than the nanocarrier drug delivery system (He et al., 2012), which lead to the less total drug in VUMON group at 2h. TEN nanosuspensions, as a passive targeted drug delivery system, permeated into tumors and got trapped due to EPR effect, and distributed into the tissues like liver, spleen, and lung, where phagocytosis cells are rich (Stylianopoulos, 2013). The larger accumulation in the tumor (Fig. 6) could be used to explain the better antitumor effect by using the drug nanosuspensions in the antitumor effect study. Orthotopic brain tumor model was better to evaluate TEN nanosuspensions. Unfortunately, due to technical reasons, orthotopic brain tumor model was not established and the drug concentrations in the tumor and other major organs were not detected at the longer time points, such as 4 h, 8 h.
4. Conclusions
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In this study, a novel teniposide formulation for intravenous administration, TEN nanosuspensions, was proposed and successfully prepared via the anti-solvent precipitation-sonication technique, PVP as the stabilizer. With the optimized formulation, TEN nanosuspensions with a mean particle size of 151 ± 11 nm and a narrow PDI 0.138 were obtained. And TEN nanosuspensions were rod-like under SEM. The obtained TEN nanosuspensions was physically and chemically stable at least 10 days at 4°C. And the lyophilized preparations were stable during 3 months. In vitro cytotoxicity study showed that TEN nanosuspensions could induce similar, if not better, cell growth inhibition for U87MG and C6, compared with VUMON. While, the in vivo antitumor test in C6 tumors bearing rats demonstrated that the inhibition rates were 69.2% and 57.2% for TEN nanosuspensions and VUMON relative to 5 % glucose group, respectively. The antitumor effect of TEN nanosuspensions was markedly superior to VUMON (p
