Colloids and Surfaces B: Biointerfaces 123 (2014) 716–723
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enhanced antitumor efficacy of vitamin E TPGS-emulsified PLGA nanoparticles for delivery of paclitaxel Yanbin Sun a,1 , Bo Yu b,1 , Guoying Wang c , Yongsheng Wu d , Xiaomin Zhang b , Yanmin Chen d , Suoqing Tang e , Yuan Yuan c , Robert J. Lee f,g , Lesheng Teng f,∗ , Shun Xu a,∗∗ a
Department of Thoracic Surgery, First Hospital, China Medical University, Shenyang, Liaoning, PR China Hangzhou PushiKang Biotechnology Co., Ltd, Zhejiang, PR China c The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China d Mudanjiang Youbo Pharmaceutical Co., Ltd, Beijing, PR China e Department of Pediatrics, PLA General Hospital, Beijing, PR China f School of Life Sciences, Jinlin University, Changchun, PR China g College of Pharmacy, The Ohio State University, Columbus, OH, USA b
a r t i c l e
i n f o
Article history: Received 30 March 2014 Received in revised form 30 September 2014 Accepted 5 October 2014 Available online 12 October 2014 Keywords: Paclitaxel TPGS Nanoparticles Lung cancer Drug delivery Chemotherapy
a b s t r a c t Nanoparticles are efficient delivery vehicles for cancer therapy such as paclitaxel (PTX). In this study, we formulated PTX into PLGA polymeric nanoparticles. Vitamin E TPGS was used as an emulsifier to stabilize the nanoparticle formulation. PTX was encapsulated in TPGS-emulsified polymeric nanoparticles (TENPs) by a nanoprecipitation method in ethanol–water system. The resultant PTX-TENPs showed a very uniform particle size (∼100 nm) and high drug encapsulation (>80%). The cytotoxicity of PTX-TENPs was examined in A549 lung cancer cell line. Preferential tumor accumulation of TENPs was observed in the A549 lung cancer xenograft model. Tumor growth was significantly inhibited by intravenous injection of PTX-TENPs. Our results suggested that the modified nanoprecipitation method holds great potential for the fabrication of the PTX loaded polymeric nanoparticles. TPGS can be used in the manufacture of polymeric nanoparticles for the controlled release of PTX and other anti-cancer drugs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel (PTX) is widely used in clinical treatment of many carcinomas such as breast cancer, advanced ovarian cancer, lung cancer, head and neck cancer and acute leukemia [1]. PTX in the clinic is formulated in a 1:1 mixture of Cremophor EL (polyethoxylated castor oil) and ethanol, and is known as Taxol® . However, Cremophor EL has been shown to cause severe side effects, such as hypersensitivity, neurotoxicity, and nephropathy [2,3]. To reduce the adverse effects caused by Cremophor EL and nonspecific delivery of PTX itself, substantial efforts have been focused on developing various drug delivery systems such as polymeric
∗ Corresponding author at: School of Life Sciences, Jinlin University, No. 2699, Qianjin Avenue, Changchun, Jilin Province, PR China. Tel.: +86 431 85168646; fax: +86 431 85168637. ∗∗ Corresponding author. E-mail address: [email protected] (L. Teng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.10.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
nanoparticles, liposomes, micelles and emulsions [4–7]. Another major problem encountered by PTX in the clinic is the development of drug resistance. Various mechanisms at the cellular level may contribute to drug resistance, including the presence of drug efflux proteins such as the p-glycoprotein (P-gp) on the cell membrane [8]. Polymeric nanoparticles (NPs) based on biodegradable polyesters, have been investigated extensively as nanocarriers for anticancer drugs [9–11]. Particular attention has been given to poly(d,l lactide-co-glycolide) (PLGA) because of its high stability, drug solubilization efficiency, biocompatibility, and passive targeting capability by the enhanced permeability and retention (EPR) effect [10,12–14]. PLGA has been approved by US FDA as a safe pharmaceutical excipient for biomedical devices and microsphere formulations. Several PLGA based nano-formulations are under clinical investigation for cancer treatment [12,15]. It has been shown that PLGA nanoparticles encapsulating anticancer drugs can enhance their intracellular concentration and overcome P-gp mediated drug resistance [16,17]. Therefore, PLGA nanoparticle mediated delivery is a promising approach for PTX.
Y. Sun et al. / Colloids and Surfaces B: Biointerfaces 123 (2014) 716–723
Anticancer drug loaded polymeric nanoparticles are usually produced by two classical methods: nanoprecipitation and emulsion-solvent evaporation. It is well accepted that the emulsifier plays a key role in the process of nanoparticle formation [18–20]. The nonionic surfactants Poloxamer 188, polyethylene glycol (1000, 2000, 4000), F68, and water-soluble vitamin E TPGS, also known as TPGS (D-␣-tocopherol polyethylene glycol 1000 succinate) are commonly used as emulsifiers in nanoparticle preparation [21–23]. On the other hand, the emulsifiers have an impact on the morphological, physicochemical and pharmaceutical properties of the produced nanoparticles. Due to its high emulsification capacity, TPGS has been used in the preparation of polymeric nanoparticles [18,24–26]. Recently, we developed an inverse-phase nanoprecipitation method to prepare TPGS-stabilized PLGA nanoparticles as a delivery system for PTX [27]. In this work, a modified nanoprecipitation method in an ethanol–water system was developed to prepare TPGSemulsified polymeric nanoparticles (TENPs). The emulsifying effect of TPGS on the nanoparticle formation was examined. PTX encapsulated TENPs (PTX-TENPs) with high drug loading efficiency and uniform size were successfully prepared. A possible mechanism of formation of PTX-TENPs was proposed. The morphology, drug release kinetics and in vitro cytotoxicity of PTX-TENPs were characterized. Furthermore, in vivo biodistribution and antitumor efficacy were also evaluated for the PTX-TENPs.
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2.3. Characterization of PTX-TENPs Mean particle size and size distribution were measured by the dynamic light scattering method using a Nicomp Zeta Potential/Particle Size (model 380XLS, NicompTM , Santa Barbara, CA, USA). The analyses were performed with 5 mW He–Ne laser (632.8 nm) at a scattering angle of 90◦ at 25 ◦ C. Each sample was placed into a quartz cuvette and diluted to the appropriate concentration. The data was presented as mean size ± SD based on three separate experiments. TEM images were recorded using a JEOL JEM-200CX instrument (TEM) at an acceleration voltage of 200 kV. The sample was prepared by administering the PTX-TENPs suspension (2 mg/mL) onto a 100-mesh formvar-coated copper grid. 2.4. Encapsulation efficiency of PTX-TENPs The concentration of PTX was determined by HPLC, which was carried out with a Chromatograph of Agilent 1200 (Agilent Technologies Inc, Cotati, CA) using Phenomenex C18 column (250 mm × 4.6 mm, 5 m) and the mobile phase of acetonitrile and water (55/45, v/v) at a flow-rate of 1 mL/min. The chromatography was carried out at 40 ◦ C. The injection volume was 20 L and the detection wavelength was 227 nm. The encapsulation efficiency (EE%) of Paclitaxel NPs was calculated as follows: EE% =
Paclitaxel weight measured in NPs × 100% Paclitaxel weight added
2. Materials and methods 2.1. Materials
2.5. In vitro release study
Poly(lactic-co-glycolic acid) (PLGA) with a 50:50 monomer ratio, ester-terminated, was ordered from DaiGang Biotechnology Co., Ltd (Jinan, China). Taxol® and paclitaxel were purchased from Bristol-Myers Squibb (Italy) and Knowshine Pharmachemicals Inc. (Shanghai, China), respectively. DiR was purchased from Caliper Life Sciences (Hopkinton, MA). The HPLC-grade solvents used for high-performance liquid chromatography (HPLC) were purchased from Sigma–Aldrich (Shanghai Local Agent, China). All other chemicals utilized were of analytical grade. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma–Aldrich (Shanghai local agent, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies).
In vitro release of PTX was studied in 0.02 mol/L phosphatebuffered saline (PBS, pH 7.4) with 0.5% Tween 80. In a dialysis tubing, approximately PTX-TENPs and Taxol® equivalent to 0.5 mg PTX were suspended in 5 mL distilled water. Then, the dialysis tubing was placed into 150 mL PBS at 37 ◦ C, followed by continuous shaking at 200 rpm and 37 ◦ C in a constant temperature shaking bath. Samples (1 mL) were withdrawn at predetermined time intervals and centrifuged. The supernatant was collected for HPLC analysis at 27 nm after passing through a 0.45 m filter. The release rate was calculated according to the equation: RR% = (Wi /Wtotal ) × 100%, where Wi is the measured amount of PTX at the indicated time point and Wtotal is the total PTX amount in the same volume of NPs suspensions. 2.6. Cell culture
2.2. Preparation of PTX-loaded TENPs PTX loaded TENPs (PTX-TENPs) were prepared by a modified nanoprecipitation method in ethanol–water system [28]. Briefly, PTX (2 mg) and the PLGA (20 mg) were dissolved in acetone (5 mL) as the organic phase. Using a syringe pump, the organic phase was added dropwise (2 mL/min) into 10 mL of a water/ethanol solution (1:1, v/v) containing 0.6% (w/v) TPGS under magnetic stirring. The organic solvents (ethanol and acetone) were then evaporated off from the resultant milky colloidal suspension under high vacuum at 40 ◦ C. The remaining organic solvent and free molecules were removed by washing the NPs three times using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cut-off of 10 kDa. After washing, the PTX-TENPs were resuspended in 5 mL distilled water. The NPs were used immediately or stored at −4 ◦ C for later use. The same procedure was used for the synthesis of fluorescent DiR loaded TENPs, except that PTX was replaced by 0.05 wt% of DiR.
A549 cells were cultured in 37.5 cm2 flasks with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 ◦ C in a humidified incubator containing 5% CO2 . 2.7. In vitro cytotoxic activity The effect of Taxol and PTX-TENPs on the viability of A549 cell was determined by the colorimetric MTT assay. The A549 cells were seeded into 96-well culture plates at 5000 cells/well and cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 for 24 h. Fresh culture media was added to the cells and the plate was incubated at 37 ◦ C for an additional 44 h before further testing. Cell viability was analyzed by MTT assay. Briefly, 20 L MTT solution was added to each well and the plate was incubated for 1 h at 37 ◦ C. Optical density was determined at 490 nm on a standard plate reader. Results were reported as a percentage of cell viability relative to the untreated control.
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Fig. 1. Characterization of the prepared PTX-TENPs. (A) A representative profile of particles size and size distribution; (B) transmission electron microscopy (TEM) images.
2.8. In vivo imaging analysis Female nude mice (16–18 g) were subcutaneously inoculated on day 0 with 1.0 × 107 cells and then randomized to different treatment groups to avoid cage effects. When a tumor reached a volume of ∼500 mm3 , 3 mice were injected intravenously with DiR-TENPs (2 mg/kg, i.v.). The mice were anesthetized and imaged at 2 h, 8 h and 24 h using a Kodak multimodal imaging system (Carestream Health, Inc., USA). The excitation bandpass filter was at 740 nm and the emission was at 790 nm. Exposure time was 5 s for each image. After in vivo imaging, the mice were sacrificed. Major organs including hearts, livers, spleens, lungs, kidneys and tumors were excised. The near-infrared fluorescence signal intensities in different tissues were measured. 2.9. Antitumor efficacy in A549 lung cancer xenograft Female BALB/c nude mice (16–18 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Ten million A549 cells were injected into each mouse subcutaneously. When tumor reached a volume of 100–150 mm3 , these mice were randomly divided into three groups (n = 8). Mice were administered intravenously with PBS, Taxol® and T45K ENPs. The Taxol® and T45K ENPs groups were administered at the equivalent PTX doses (10 mg/kg) for comparison. The mice were then monitored
for tumor progression and weight loss every three days. Tumor volumes were calculated by length × width2 /2 (mm3 ). The tumor volume inhibitory rate at day 24 was calculated with the formula: Rv = 1 − (Vdrug /VPBS ) × 100%, where Vdrug is the tumor volume after treatment, and VPBS is the tumor volume after treatment with PBS. Changes in body weight of each mouse were also monitored during the treatment to evaluate possible toxic effects of the therapy. 3. Results 3.1. Preparation and characterization of the PTX-TENPs We adopted a nanoprecipitation method to prepare PTX loaded PLGA nanoparticles. TPGS was used as an emulsifier to facilitate nanoparticle formation in an ethanol–water system. In the preparing process, the organic phase containing PLGA and PTX was slowly added into the aqueous phase containing TPGS. With slowly stirring, the organic phase was gradually dispersed in aqueous phase. When the organic solvents were evaporated off, PTX was enveloped by PLGA and precipitated to form nanoparticle. TPGS was simultaneously coated around PLGA polymeric core. The representative size distribution profile and morphology of PTX-TENPs are shown in Fig. 1. It can be seen that the average size of PTX-TENPs was about 120 nm with a low PDI ∼0.1 (Fig. 1A). The zeta potential of PTX-TENPs was between −15 mV and −22 mV. The
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Fig. 2. The influence factors on the properties of nanoparticle. Effect of the TPGS/PLGA polymer weight ratio on the nanoparticle size and PDI (A) and the EE% (B); effect of PLGA molecular weights on the nanoparticle size and PDI (C) and the EE% (D) at a fixed TPGS/PLGA polymer weight ratio 3. The drug EE% was determined by HPLC.
morphology of PTX-TENPs was further characterized by TEMs. The TEM images in Fig. 1B revealed that the PTX-TENPs were dispersed with a well-defined spherical shape around 120 nm in diameter, which is similar to the results obtained by the DLS technique (Fig. 1A). The surrounding shadows of TENPs were phosphotungstic acid, which was used for a stronger contrast between the polymeric biomaterials and the background. 3.2. Optimization of the PTX-TENPs The effects of some factors on the particle size, zeta potential and EE were investigated for PTX-TENPs formation. First, by fixing the final aqueous volume to organic solution ratio of 3:1, we examined the effect of the mass ratio of TPGS to PLGA (from 1 to 6). As shown in Fig. 2A, the nanoparticle size slightly increases with the increase of TPGS/PLGA mass ratio. The smallest size was achieved at the mass ratio of 2. The more TPGS in the formulation led to the lower PDI. Fig. 2B revealed that with the increasing of TPGS/PLGA mass ratio, the EE% of PTX improved. The EE% remained almost unchanged after the mass ratio achieved 3. These results suggest that the sufficient TPGS can stabilize the PTX-TENPs and improve EE% of PTX and the optimal mass ratio of TPGS/PLGA is 3. Next, at a fixed TPGS/PLGA mass ratio of 3, we further investigated the effect of molecular weight of PLGA on the PTX-TENPs
properties. Four different molecular weights of PLGA (15 K, 45 K, 60 K and 100 K) were chosen. The resultant PTX-TENPs were named T15K ENPs, T45K ENPs, T60K ENPs and T100K ENPs, respectively. Fig. 2C showed that the smallest size and PDI can be achieved when the PLGA with the molecular weight of 45 K was chosen. When the molecular weight of PLGA was higher than 45 K, it seems that varying the molecular weight of PLGA did not cause obvious changes in particle size. However, the PDI increased with the increase of PLGA molecular weights. As shown in Fig. 2D, the molecular weight of PLGA had the slight impact on the EE% of PTX. Thus, we selected TENPs with the TPGS/PLGA mass ratio of 3 and with PLGA molecular weight of 45 K for the subsequent nanoparticle preparation unless otherwise noticed. 3.3. In vitro drug release The drug release profiles of the PTX-TENPs in vitro are shown in Fig. 3. T45K ENPs and T100K ENPs had a similar release profile of PTX; approximate 25% of PTX was released at a constant rate during the first 24 h. After that, a slower release rate was observed; nearly 50% PTX was released at 96 h. Compared with the release profile of T45K ENPs and T100K ENPs, T15K ENPs exhibited a much faster release rate. Approximately 25% of incorporated PTX was rapidly released from PTX-TENPs during the initial period of 12 h.
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Fig. 3. In vitro accumulative release profiles of PTX-TENPs. Effect of the molecular weight of PLGA on the PTX release of TENPs was examined. Data are presented as the mean ± standard deviation (SD).
The release reached 55% at the time point of 36 h. Afterwards, the release of PTX slowed and 70% of PTX was released cumulatively at 96 h. 3.4. In vitro cytotoxicity As shown in Fig. 4, the concentration-dependent cell growth inhibition activity of both Taxol and T45K ENPs was
Fig. 4. In vitro cytotoxicity study. Cell viability of A549 cells as a function of varying concentrations of PTX at 48 h. Each point represents mean ± SD (n = 4).
observed on the A549 cells. At high drug concentrations, significant greater cell viabilities reduction was observed for the PTX-T45K ENPs treated group, in comparison with that on the Taxol® at the same concentration. The blank T45K ENPs had certain inhibitory activity on the tumor cells after 48 h incubation.
Fig. 5. The tumor accumulation study of TENPs. (A) The non-invasive IVIS images of time-dependent whole body imaging of A549 tumor-bearing mice at 2 h, 8 h and 24 h after intravenous injection. (B) Tissue distribution of DiR-TENPs. 2 h, 8 h and 24 h after intravenous administration, tissues were harvested and then DiR fluorescence signals were measured by IVIS imaging system. (C) Tissue biodistribution based on the intensity of DiR signal (n = 4).
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the 21st day (Fig. 6). Compared to Taxol® , PTX-T45K ENPs showed superior antitumor effects. The PTX-T45K ENPs exhibited the strongest antitumor efficacy. Body weight was determined, as shown in Fig. 6B. After 21 days, the mice in the control group and the treatment group have no statistically significant differences in body weight.
4. Discussion
Fig. 6. In vivo antitumor efficacy of PTX-TENPs. The mice were intravenously administered with PBS, Taxol® (10 mg/kg), and PTX-T45K ENPs (10 mg/kg) every three days for three consecutive injections. (A) Growth inhibition study in the A549 xenograft model; (B) body weight changes for the tumor-bearing mice after various formulations were given to mice on the indicated days (shown by arrow). Data are presented as the mean ± standard deviation (n = 8).
3.5. Biodistribution study In order to determine the in vivo biodistribution of TENPs, DiR-labeled T45K ENPs were injected intravenously into A549 lung cancer tumor bearing mice through the tail vein and time-dependant biodistribution was observed using non-invasive fluorescence imaging in live animals. As shown in Fig. 5A, fluorescence signal was observed in the tumor as early as 2 h after i.v. injection and reached a maximum at 24 h post-injection. Major organs including liver, lung, kidney, spleen and heart were harvested 4 h later. The fluorescence signal of DiR-TENPs was analyzed by IVIS. Fig. 5B and C clearly showed that the TENPs had preferential tumor accumulation compared to other organs. These results indicated that the T45K NPs had strong passive tumor targeting capability through the EPR effect [10,13,27]. 3.6. Anti-tumor efficacy in A549 lung cancer xenograft model The above-mentioned favorable tumor accumulation characteristics of TENPs encouraged us to investigate the in vivo antitumor activity of PTX-TENPs in mice bearing A549 lung cancer xenograft model. The mice were intravenously administered with PBS, Taxol® , or PTX-T45K ENPs at a PTX dose of 10 mg/kg every three days for three consecutive injections. Tumors size was recorded until
Development of suitable drug delivery systems for hydrophobic drugs such as PTX is a major focus of the nanomedicine field [4,15,6]. In this study, to achieve efficient PTX delivery into tumor, PTX was encapsulated in TENPs. Polymeric nanoparticles are derived from PLGA since their safety in clinic is well established [10,12]. By this nanoprecipitation method, the prepared PTX-TENPs exhibited the well-defined spherical shape, small particle size, low PDI (∼0.1) (Fig. 1). The PTX-TENPs also possess high solubility of PTX (>1 mg/mL) and sustained drug release property (Fig. 3). The lower release rate at initial 2 h (
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enhanced antitumor efficacy of vitamin E TPGS-emulsified PLGA nanoparticles for delivery of paclitaxel Yanbin Sun a,1 , Bo Yu b,1 , Guoying Wang c , Yongsheng Wu d , Xiaomin Zhang b , Yanmin Chen d , Suoqing Tang e , Yuan Yuan c , Robert J. Lee f,g , Lesheng Teng f,∗ , Shun Xu a,∗∗ a
Department of Thoracic Surgery, First Hospital, China Medical University, Shenyang, Liaoning, PR China Hangzhou PushiKang Biotechnology Co., Ltd, Zhejiang, PR China c The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China d Mudanjiang Youbo Pharmaceutical Co., Ltd, Beijing, PR China e Department of Pediatrics, PLA General Hospital, Beijing, PR China f School of Life Sciences, Jinlin University, Changchun, PR China g College of Pharmacy, The Ohio State University, Columbus, OH, USA b
a r t i c l e
i n f o
Article history: Received 30 March 2014 Received in revised form 30 September 2014 Accepted 5 October 2014 Available online 12 October 2014 Keywords: Paclitaxel TPGS Nanoparticles Lung cancer Drug delivery Chemotherapy
a b s t r a c t Nanoparticles are efficient delivery vehicles for cancer therapy such as paclitaxel (PTX). In this study, we formulated PTX into PLGA polymeric nanoparticles. Vitamin E TPGS was used as an emulsifier to stabilize the nanoparticle formulation. PTX was encapsulated in TPGS-emulsified polymeric nanoparticles (TENPs) by a nanoprecipitation method in ethanol–water system. The resultant PTX-TENPs showed a very uniform particle size (∼100 nm) and high drug encapsulation (>80%). The cytotoxicity of PTX-TENPs was examined in A549 lung cancer cell line. Preferential tumor accumulation of TENPs was observed in the A549 lung cancer xenograft model. Tumor growth was significantly inhibited by intravenous injection of PTX-TENPs. Our results suggested that the modified nanoprecipitation method holds great potential for the fabrication of the PTX loaded polymeric nanoparticles. TPGS can be used in the manufacture of polymeric nanoparticles for the controlled release of PTX and other anti-cancer drugs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel (PTX) is widely used in clinical treatment of many carcinomas such as breast cancer, advanced ovarian cancer, lung cancer, head and neck cancer and acute leukemia [1]. PTX in the clinic is formulated in a 1:1 mixture of Cremophor EL (polyethoxylated castor oil) and ethanol, and is known as Taxol® . However, Cremophor EL has been shown to cause severe side effects, such as hypersensitivity, neurotoxicity, and nephropathy [2,3]. To reduce the adverse effects caused by Cremophor EL and nonspecific delivery of PTX itself, substantial efforts have been focused on developing various drug delivery systems such as polymeric
∗ Corresponding author at: School of Life Sciences, Jinlin University, No. 2699, Qianjin Avenue, Changchun, Jilin Province, PR China. Tel.: +86 431 85168646; fax: +86 431 85168637. ∗∗ Corresponding author. E-mail address: [email protected] (L. Teng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.10.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
nanoparticles, liposomes, micelles and emulsions [4–7]. Another major problem encountered by PTX in the clinic is the development of drug resistance. Various mechanisms at the cellular level may contribute to drug resistance, including the presence of drug efflux proteins such as the p-glycoprotein (P-gp) on the cell membrane [8]. Polymeric nanoparticles (NPs) based on biodegradable polyesters, have been investigated extensively as nanocarriers for anticancer drugs [9–11]. Particular attention has been given to poly(d,l lactide-co-glycolide) (PLGA) because of its high stability, drug solubilization efficiency, biocompatibility, and passive targeting capability by the enhanced permeability and retention (EPR) effect [10,12–14]. PLGA has been approved by US FDA as a safe pharmaceutical excipient for biomedical devices and microsphere formulations. Several PLGA based nano-formulations are under clinical investigation for cancer treatment [12,15]. It has been shown that PLGA nanoparticles encapsulating anticancer drugs can enhance their intracellular concentration and overcome P-gp mediated drug resistance [16,17]. Therefore, PLGA nanoparticle mediated delivery is a promising approach for PTX.
Y. Sun et al. / Colloids and Surfaces B: Biointerfaces 123 (2014) 716–723
Anticancer drug loaded polymeric nanoparticles are usually produced by two classical methods: nanoprecipitation and emulsion-solvent evaporation. It is well accepted that the emulsifier plays a key role in the process of nanoparticle formation [18–20]. The nonionic surfactants Poloxamer 188, polyethylene glycol (1000, 2000, 4000), F68, and water-soluble vitamin E TPGS, also known as TPGS (D-␣-tocopherol polyethylene glycol 1000 succinate) are commonly used as emulsifiers in nanoparticle preparation [21–23]. On the other hand, the emulsifiers have an impact on the morphological, physicochemical and pharmaceutical properties of the produced nanoparticles. Due to its high emulsification capacity, TPGS has been used in the preparation of polymeric nanoparticles [18,24–26]. Recently, we developed an inverse-phase nanoprecipitation method to prepare TPGS-stabilized PLGA nanoparticles as a delivery system for PTX [27]. In this work, a modified nanoprecipitation method in an ethanol–water system was developed to prepare TPGSemulsified polymeric nanoparticles (TENPs). The emulsifying effect of TPGS on the nanoparticle formation was examined. PTX encapsulated TENPs (PTX-TENPs) with high drug loading efficiency and uniform size were successfully prepared. A possible mechanism of formation of PTX-TENPs was proposed. The morphology, drug release kinetics and in vitro cytotoxicity of PTX-TENPs were characterized. Furthermore, in vivo biodistribution and antitumor efficacy were also evaluated for the PTX-TENPs.
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2.3. Characterization of PTX-TENPs Mean particle size and size distribution were measured by the dynamic light scattering method using a Nicomp Zeta Potential/Particle Size (model 380XLS, NicompTM , Santa Barbara, CA, USA). The analyses were performed with 5 mW He–Ne laser (632.8 nm) at a scattering angle of 90◦ at 25 ◦ C. Each sample was placed into a quartz cuvette and diluted to the appropriate concentration. The data was presented as mean size ± SD based on three separate experiments. TEM images were recorded using a JEOL JEM-200CX instrument (TEM) at an acceleration voltage of 200 kV. The sample was prepared by administering the PTX-TENPs suspension (2 mg/mL) onto a 100-mesh formvar-coated copper grid. 2.4. Encapsulation efficiency of PTX-TENPs The concentration of PTX was determined by HPLC, which was carried out with a Chromatograph of Agilent 1200 (Agilent Technologies Inc, Cotati, CA) using Phenomenex C18 column (250 mm × 4.6 mm, 5 m) and the mobile phase of acetonitrile and water (55/45, v/v) at a flow-rate of 1 mL/min. The chromatography was carried out at 40 ◦ C. The injection volume was 20 L and the detection wavelength was 227 nm. The encapsulation efficiency (EE%) of Paclitaxel NPs was calculated as follows: EE% =
Paclitaxel weight measured in NPs × 100% Paclitaxel weight added
2. Materials and methods 2.1. Materials
2.5. In vitro release study
Poly(lactic-co-glycolic acid) (PLGA) with a 50:50 monomer ratio, ester-terminated, was ordered from DaiGang Biotechnology Co., Ltd (Jinan, China). Taxol® and paclitaxel were purchased from Bristol-Myers Squibb (Italy) and Knowshine Pharmachemicals Inc. (Shanghai, China), respectively. DiR was purchased from Caliper Life Sciences (Hopkinton, MA). The HPLC-grade solvents used for high-performance liquid chromatography (HPLC) were purchased from Sigma–Aldrich (Shanghai Local Agent, China). All other chemicals utilized were of analytical grade. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma–Aldrich (Shanghai local agent, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies).
In vitro release of PTX was studied in 0.02 mol/L phosphatebuffered saline (PBS, pH 7.4) with 0.5% Tween 80. In a dialysis tubing, approximately PTX-TENPs and Taxol® equivalent to 0.5 mg PTX were suspended in 5 mL distilled water. Then, the dialysis tubing was placed into 150 mL PBS at 37 ◦ C, followed by continuous shaking at 200 rpm and 37 ◦ C in a constant temperature shaking bath. Samples (1 mL) were withdrawn at predetermined time intervals and centrifuged. The supernatant was collected for HPLC analysis at 27 nm after passing through a 0.45 m filter. The release rate was calculated according to the equation: RR% = (Wi /Wtotal ) × 100%, where Wi is the measured amount of PTX at the indicated time point and Wtotal is the total PTX amount in the same volume of NPs suspensions. 2.6. Cell culture
2.2. Preparation of PTX-loaded TENPs PTX loaded TENPs (PTX-TENPs) were prepared by a modified nanoprecipitation method in ethanol–water system [28]. Briefly, PTX (2 mg) and the PLGA (20 mg) were dissolved in acetone (5 mL) as the organic phase. Using a syringe pump, the organic phase was added dropwise (2 mL/min) into 10 mL of a water/ethanol solution (1:1, v/v) containing 0.6% (w/v) TPGS under magnetic stirring. The organic solvents (ethanol and acetone) were then evaporated off from the resultant milky colloidal suspension under high vacuum at 40 ◦ C. The remaining organic solvent and free molecules were removed by washing the NPs three times using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cut-off of 10 kDa. After washing, the PTX-TENPs were resuspended in 5 mL distilled water. The NPs were used immediately or stored at −4 ◦ C for later use. The same procedure was used for the synthesis of fluorescent DiR loaded TENPs, except that PTX was replaced by 0.05 wt% of DiR.
A549 cells were cultured in 37.5 cm2 flasks with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 ◦ C in a humidified incubator containing 5% CO2 . 2.7. In vitro cytotoxic activity The effect of Taxol and PTX-TENPs on the viability of A549 cell was determined by the colorimetric MTT assay. The A549 cells were seeded into 96-well culture plates at 5000 cells/well and cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 for 24 h. Fresh culture media was added to the cells and the plate was incubated at 37 ◦ C for an additional 44 h before further testing. Cell viability was analyzed by MTT assay. Briefly, 20 L MTT solution was added to each well and the plate was incubated for 1 h at 37 ◦ C. Optical density was determined at 490 nm on a standard plate reader. Results were reported as a percentage of cell viability relative to the untreated control.
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Fig. 1. Characterization of the prepared PTX-TENPs. (A) A representative profile of particles size and size distribution; (B) transmission electron microscopy (TEM) images.
2.8. In vivo imaging analysis Female nude mice (16–18 g) were subcutaneously inoculated on day 0 with 1.0 × 107 cells and then randomized to different treatment groups to avoid cage effects. When a tumor reached a volume of ∼500 mm3 , 3 mice were injected intravenously with DiR-TENPs (2 mg/kg, i.v.). The mice were anesthetized and imaged at 2 h, 8 h and 24 h using a Kodak multimodal imaging system (Carestream Health, Inc., USA). The excitation bandpass filter was at 740 nm and the emission was at 790 nm. Exposure time was 5 s for each image. After in vivo imaging, the mice were sacrificed. Major organs including hearts, livers, spleens, lungs, kidneys and tumors were excised. The near-infrared fluorescence signal intensities in different tissues were measured. 2.9. Antitumor efficacy in A549 lung cancer xenograft Female BALB/c nude mice (16–18 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Ten million A549 cells were injected into each mouse subcutaneously. When tumor reached a volume of 100–150 mm3 , these mice were randomly divided into three groups (n = 8). Mice were administered intravenously with PBS, Taxol® and T45K ENPs. The Taxol® and T45K ENPs groups were administered at the equivalent PTX doses (10 mg/kg) for comparison. The mice were then monitored
for tumor progression and weight loss every three days. Tumor volumes were calculated by length × width2 /2 (mm3 ). The tumor volume inhibitory rate at day 24 was calculated with the formula: Rv = 1 − (Vdrug /VPBS ) × 100%, where Vdrug is the tumor volume after treatment, and VPBS is the tumor volume after treatment with PBS. Changes in body weight of each mouse were also monitored during the treatment to evaluate possible toxic effects of the therapy. 3. Results 3.1. Preparation and characterization of the PTX-TENPs We adopted a nanoprecipitation method to prepare PTX loaded PLGA nanoparticles. TPGS was used as an emulsifier to facilitate nanoparticle formation in an ethanol–water system. In the preparing process, the organic phase containing PLGA and PTX was slowly added into the aqueous phase containing TPGS. With slowly stirring, the organic phase was gradually dispersed in aqueous phase. When the organic solvents were evaporated off, PTX was enveloped by PLGA and precipitated to form nanoparticle. TPGS was simultaneously coated around PLGA polymeric core. The representative size distribution profile and morphology of PTX-TENPs are shown in Fig. 1. It can be seen that the average size of PTX-TENPs was about 120 nm with a low PDI ∼0.1 (Fig. 1A). The zeta potential of PTX-TENPs was between −15 mV and −22 mV. The
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Fig. 2. The influence factors on the properties of nanoparticle. Effect of the TPGS/PLGA polymer weight ratio on the nanoparticle size and PDI (A) and the EE% (B); effect of PLGA molecular weights on the nanoparticle size and PDI (C) and the EE% (D) at a fixed TPGS/PLGA polymer weight ratio 3. The drug EE% was determined by HPLC.
morphology of PTX-TENPs was further characterized by TEMs. The TEM images in Fig. 1B revealed that the PTX-TENPs were dispersed with a well-defined spherical shape around 120 nm in diameter, which is similar to the results obtained by the DLS technique (Fig. 1A). The surrounding shadows of TENPs were phosphotungstic acid, which was used for a stronger contrast between the polymeric biomaterials and the background. 3.2. Optimization of the PTX-TENPs The effects of some factors on the particle size, zeta potential and EE were investigated for PTX-TENPs formation. First, by fixing the final aqueous volume to organic solution ratio of 3:1, we examined the effect of the mass ratio of TPGS to PLGA (from 1 to 6). As shown in Fig. 2A, the nanoparticle size slightly increases with the increase of TPGS/PLGA mass ratio. The smallest size was achieved at the mass ratio of 2. The more TPGS in the formulation led to the lower PDI. Fig. 2B revealed that with the increasing of TPGS/PLGA mass ratio, the EE% of PTX improved. The EE% remained almost unchanged after the mass ratio achieved 3. These results suggest that the sufficient TPGS can stabilize the PTX-TENPs and improve EE% of PTX and the optimal mass ratio of TPGS/PLGA is 3. Next, at a fixed TPGS/PLGA mass ratio of 3, we further investigated the effect of molecular weight of PLGA on the PTX-TENPs
properties. Four different molecular weights of PLGA (15 K, 45 K, 60 K and 100 K) were chosen. The resultant PTX-TENPs were named T15K ENPs, T45K ENPs, T60K ENPs and T100K ENPs, respectively. Fig. 2C showed that the smallest size and PDI can be achieved when the PLGA with the molecular weight of 45 K was chosen. When the molecular weight of PLGA was higher than 45 K, it seems that varying the molecular weight of PLGA did not cause obvious changes in particle size. However, the PDI increased with the increase of PLGA molecular weights. As shown in Fig. 2D, the molecular weight of PLGA had the slight impact on the EE% of PTX. Thus, we selected TENPs with the TPGS/PLGA mass ratio of 3 and with PLGA molecular weight of 45 K for the subsequent nanoparticle preparation unless otherwise noticed. 3.3. In vitro drug release The drug release profiles of the PTX-TENPs in vitro are shown in Fig. 3. T45K ENPs and T100K ENPs had a similar release profile of PTX; approximate 25% of PTX was released at a constant rate during the first 24 h. After that, a slower release rate was observed; nearly 50% PTX was released at 96 h. Compared with the release profile of T45K ENPs and T100K ENPs, T15K ENPs exhibited a much faster release rate. Approximately 25% of incorporated PTX was rapidly released from PTX-TENPs during the initial period of 12 h.
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Fig. 3. In vitro accumulative release profiles of PTX-TENPs. Effect of the molecular weight of PLGA on the PTX release of TENPs was examined. Data are presented as the mean ± standard deviation (SD).
The release reached 55% at the time point of 36 h. Afterwards, the release of PTX slowed and 70% of PTX was released cumulatively at 96 h. 3.4. In vitro cytotoxicity As shown in Fig. 4, the concentration-dependent cell growth inhibition activity of both Taxol and T45K ENPs was
Fig. 4. In vitro cytotoxicity study. Cell viability of A549 cells as a function of varying concentrations of PTX at 48 h. Each point represents mean ± SD (n = 4).
observed on the A549 cells. At high drug concentrations, significant greater cell viabilities reduction was observed for the PTX-T45K ENPs treated group, in comparison with that on the Taxol® at the same concentration. The blank T45K ENPs had certain inhibitory activity on the tumor cells after 48 h incubation.
Fig. 5. The tumor accumulation study of TENPs. (A) The non-invasive IVIS images of time-dependent whole body imaging of A549 tumor-bearing mice at 2 h, 8 h and 24 h after intravenous injection. (B) Tissue distribution of DiR-TENPs. 2 h, 8 h and 24 h after intravenous administration, tissues were harvested and then DiR fluorescence signals were measured by IVIS imaging system. (C) Tissue biodistribution based on the intensity of DiR signal (n = 4).
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the 21st day (Fig. 6). Compared to Taxol® , PTX-T45K ENPs showed superior antitumor effects. The PTX-T45K ENPs exhibited the strongest antitumor efficacy. Body weight was determined, as shown in Fig. 6B. After 21 days, the mice in the control group and the treatment group have no statistically significant differences in body weight.
4. Discussion
Fig. 6. In vivo antitumor efficacy of PTX-TENPs. The mice were intravenously administered with PBS, Taxol® (10 mg/kg), and PTX-T45K ENPs (10 mg/kg) every three days for three consecutive injections. (A) Growth inhibition study in the A549 xenograft model; (B) body weight changes for the tumor-bearing mice after various formulations were given to mice on the indicated days (shown by arrow). Data are presented as the mean ± standard deviation (n = 8).
3.5. Biodistribution study In order to determine the in vivo biodistribution of TENPs, DiR-labeled T45K ENPs were injected intravenously into A549 lung cancer tumor bearing mice through the tail vein and time-dependant biodistribution was observed using non-invasive fluorescence imaging in live animals. As shown in Fig. 5A, fluorescence signal was observed in the tumor as early as 2 h after i.v. injection and reached a maximum at 24 h post-injection. Major organs including liver, lung, kidney, spleen and heart were harvested 4 h later. The fluorescence signal of DiR-TENPs was analyzed by IVIS. Fig. 5B and C clearly showed that the TENPs had preferential tumor accumulation compared to other organs. These results indicated that the T45K NPs had strong passive tumor targeting capability through the EPR effect [10,13,27]. 3.6. Anti-tumor efficacy in A549 lung cancer xenograft model The above-mentioned favorable tumor accumulation characteristics of TENPs encouraged us to investigate the in vivo antitumor activity of PTX-TENPs in mice bearing A549 lung cancer xenograft model. The mice were intravenously administered with PBS, Taxol® , or PTX-T45K ENPs at a PTX dose of 10 mg/kg every three days for three consecutive injections. Tumors size was recorded until
Development of suitable drug delivery systems for hydrophobic drugs such as PTX is a major focus of the nanomedicine field [4,15,6]. In this study, to achieve efficient PTX delivery into tumor, PTX was encapsulated in TENPs. Polymeric nanoparticles are derived from PLGA since their safety in clinic is well established [10,12]. By this nanoprecipitation method, the prepared PTX-TENPs exhibited the well-defined spherical shape, small particle size, low PDI (∼0.1) (Fig. 1). The PTX-TENPs also possess high solubility of PTX (>1 mg/mL) and sustained drug release property (Fig. 3). The lower release rate at initial 2 h (
