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Biodistribution and in vivo performance of fattigation-platform theranostic nanoparticles
Materials Science and Engineering C 79 (2017) 671–678
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biodistribution and in vivo performance of fattigation-platform theranostic nanoparticles Thao Truong-Dinh Tran a, Phuong Ha-Lien Tran b, Hardik H. Amin c, Beom-Jin Lee c,⁎ a b c
Pharmaceutical Engineering Laboratory, Biomedical Engineering Department, International University, Vietnam National University – Ho Chi Minh City, 700000, Vietnam School of Medicine, Deakin University, Geelong, Australia Bioavailability Control Laboratory, College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea
a r t i c l e
i n f o
Article history: Received 23 March 2017 Received in revised form 25 April 2017 Accepted 7 May 2017 Available online 8 May 2017 Keywords: Paclitaxel Surface-functionalized magnetic nanoparticles Drug delivery Antitumor efficacy Acute toxicity
a b s t r a c t This study was aimed at characterizing superparamagnetic nanoparticles surface-functionalized with gelatinoleic acid (GOAS-MNPs) and loaded with paclitaxel by assessing the pharmacokinetics and biodistribution of paclitaxel in tissues and the in vivo efficacy of antitumor activity after the administration of the drug. Initially, instrumental analysis was performed to examine the particle size distribution, surface charge, and morphology of the paclitaxel-loaded GOAS-MNPs. Furthermore, we evaluated their magnetic properties and performed T2-weighted magnetic resonance imaging (MRI) on cells. We intravenously administered Taxol® and paclitaxel-loaded GOAS-MNPs and compared the pharmacokinetics, biodistribution, and antitumor efficacies of the two formulations. Determination of the pharmacokinetics and the biodistribution of paclitaxel-loaded NPs showed that this formulation increased the systemic circulation time of paclitaxel and regulated its transport to tissues. The in vivo antitumor efficacy of the paclitaxel-loaded NPs was better than that of Taxol® at the same dose. Furthermore, the paclitaxel-loaded GOAS-MNPs were found to be effective as contrast agents for enhanced MRI in cancer cells. Thus, GOAS-MNPs could be an effective diagnostic system for cancer and for the delivery of paclitaxel with better therapeutic effects and a significant reduction in toxicity. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is effective against various cancers and shows particularly promising results in patients with breast and ovarian cancers [1]. Because paclitaxel is highly hydrophobic and poorly soluble in aqueous media, it has a low therapeutic index [1,2]. Recently, increasing efforts have been made toward developing paclitaxel formulations for cancer treatment. Many studies are being performed to develop a paclitaxel formulation with reduced toxicity compared to that caused by Cremophor® EL. Various approaches, such as the formation of emulsions [3], liposomes [4], conjugates [5], nanoparticles (NPs) [6–10], lipid nanocapsules [1] and water-soluble prodrugs [11,12], have been investigated to increase drug efficacy and reduce the side effects of the Taxol® formulation. Recent advances in the field of nanotechnology have enabled the development of promising diagnostic agents for the imaging of health and disease conditions and for drug discovery and therapeutic delivery [13]. Especially, hybrid materials and inorganic materials are promising materials for theranostics [9,10,14–28]. Compared to conventional dosage ⁎ Corresponding author at: Bioavailability Control Laboratory, College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea. E-mail address: [email protected] (B.-J. Lee).
http://dx.doi.org/10.1016/j.msec.2017.05.029 0928-4931/© 2017 Elsevier B.V. All rights reserved.
forms, NPs have many advantages. In addition to protecting a drug from biodegradation, NPs can target the delivery of the drug to the site of action and reduce the side effects of chemotherapy [26,29,30]. The most promising application of NPs is their use in anticancer treatment. Generally, solid tumors show hypervascular permeability and impair lymphatic drainage, which creates enhanced permeability and retention (EPR) at the tumor site [31–33]. Thus, NPs show significant accumulation in the tumor and may be useful as sustained-release formulations for injection because of their small size. Furthermore, magnetic resonance imaging (MRI) is an appealing noninvasive approach for clinical diagnosis [34]. However, MRI may lack the sensitivity required to scan small tumors because of the low contrast between tumor tissue and normal tissues. Thus, it is very interesting to develop magnetic nanoparticles (MNPs) with dual roles as carriers for the delivery of anticancer drugs and as imaging contrast agents for biomedical applications. Iron oxide NPs can serve as contrast agents in MRI and enhance the detection of lesions within the body [35]. Therefore, MNPs could be used simultaneously to visualize tumors with MRI and to administer drug therapy. We have successfully synthesized surface-functionalized magnetic nanoparticles (GOAS-MNPs) in our previous studies [36]. These paclitaxel-loaded NPs had lower cytotoxicity but a similar anticancer effect as that of Taxol®. In this study, we characterized and evaluated the in
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vivo efficacy of this nanoparticle system as a novel drug-delivering MRI contrast agent. We determined the pharmacokinetic behavior, biodistribution, and in vivo antitumor activity of this system and compared them with those of Taxol®. These studies proved that these paclitaxel-loaded NPs had many advantages, such as better antitumor efficacy and increased systemic circulation time.
was purchased from J. T. Baker (Phillipsburg, USA). Oleic acid (OA) was supplied from Shinyo Pure Chemicals Co., Ltd. (Osaka, Japan). Triethylamine was purchased from Showa (Osaka, Japan). Agar was obtained from Difco Laboratories (Detroit, USA). Paclitaxel was supplied from Dae Woong Pharmaceutical Co., Ltd. (Seoul, Korea). 2.2. Methods
2. Materials and methods 2.1. Materials Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin mixtures and trypsin-EDTA were supplied from Gibco BRL (Carlsbad, CA, USA). EMT-6 and CT26 cell lines were purchased from American Type Culture Collection (ATCC, USA). Tetraethyl orthosilicate (TEOS), iron (II) chloride, iron (III) chloride, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), N1-(3-trimethoxysilylpropyl)diethylenetriamine (DETA), gelatin (GEL), 2,4,6-trinitrobenzenesulfonic acid (TNBS), 1-ethyl-3-(3dimethylamino-propyl)carbodiimide (EDC), Cremophor EL, 2-(Nmorpholino)ethanesulfonic acid (MES), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (MTT) were purchased from Sigma (St. Louis, MO, USA). Anhydrous dimethylformamide (DMF)
2.2.1. Preparation of surface-functionalized magnetic nanoparticles (GOASMNPs) GOAS-MNPs were developed in several primary steps according to the methods used in our previous studies [36]: (1) Iron oxide NPs, i.e., MNPs, were prepared by co-precipitation of FeCl3·6H2O and FeCl2·4H2O with ammonium hydroxide according to the method reported previously [37]. (2) Iron oxide NPs were added to 100 mL of absolute ethanol (0.5 mg/mL), 5 mL of water, 2.5 mL of ammonium hydroxide (28–30 wt%), and 1 mL TEOS. This mixture was allowed to react for 5 h. Then, silanization of the NPs was performed to functionalize the amines on the surface of the NPs [38]. We used 20 mL of 1% DETA in 1 mM acetic acid to disperse 30 mg of silica-coated iron oxide NPs. The amine-functionalized nanoparticles (AS-MNPs) were formed by a 30 min reaction at room
Fig. 1. Characterization of paclitaxel-loaded surface-functionalized magnetic nanoparticles (GOAS-MNPs) using scanning electron microscopy (SEM) (A), transmission electron microscopy (TEM) (B), and powder X-ray diffraction (PXRD) patterns (C).
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temperature. (3) Oleic acid (OA) was functionalized onto AS-MNPs by conjugation with the amino groups to form OAS-MNPs. OA was first dissolved in 10 mL of anhydrous DMF containing 1% triethylamine (DMFTEA) (19.75 mg/mL). This mixture was then activated using DCC and NHS (molar ratio, 1:1:1) for 30 min. Next, the ASMNPs were dispersed in DMF-TEA (200 mg/30 mL) to form a suspension, and the abovementioned activated OA solution was added and allowed to react for 2 h. (4) For conjugation of gelatin to the surface of OAS-MNPs, 50 mg of OAS-MNPs were dispersed in pH 7.5 solution and mixed with activated gelatin solution (25 mg gelatin + 50 mL pH 6 solution at 37 °C was reacted with EDC and NHS at a 1:2:2 M ratio for 30 min) for 2 h.
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(5) In each step above, the NPs were purified by using an external permanent magnet and washing with distilled water (or DMF, in the case of oleic acid conjugation) before drying under vacuum.
2.2.2. Preparation of paclitaxel-loaded NPs GOAS-MNPs (50 mg) were suspended in 20 mL of water at 37 °C. Paclitaxel was dissolved in ethanol at a concentration of 50 mg/mL. Then, 100 μL of the drug solution was added dropwise to the NP suspension. Then, this mixture was stirred to allow the absorption of the drug into the NPs. After 5 h, the drug-loaded NPs were separated using a magnet, washed several times with distilled water, and then dried in a vacuum dryer. Drug loading and encapsulation
Fig. 2. Magnetic properties of paclitaxel-loaded surface-functionalized magnetic nanoparticles (GOAS-MNPs): magnetization curve (A), T2-relaxation analysis curves (B), and T2-weighted magnetic resonance imaging (MRI) of cancer cells with nanoparticles containing different iron concentrations (C). Red indicates high signal intensity, and blue indicates low signal intensity.
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the samples were scanned in steps of 0.02° (diffraction angle, 2θ) at a rate of 1 s per step using a zero background sample holder through a D5005 diffractometer (Bruker, Germany) and Cu-Kα radiation at a voltage of 40 kV, 50 mA. 2.2.7. Magnetization measurements Measurements of the magnetization of NPs were performed at room temperature using a vibrating sample magnetometer (VSM, Lakeshore 700).
Fig. 3. Plasma curves of paclitaxel in Taxol® and drug-loaded MNPs. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
efficiency were determined indirectly by analyzing the supernatant and washings collected from the NPs. 2.2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was used to characterize surface morphology and particle shape. The samples were examined using a Cambridge Stereo Scan 200 (London, England) at an accelerating voltage of 15 kV. The samples were mounted onto brass stages using double-sided adhesive tape and coated with gold-palladium for 60 s under an argon atmosphere using a JEOL JPC-1100 sputter coater (JEOL, Japan). 2.2.4. Transmission electron microscopy Transmission electron microscopy (TEM) was used to investigate the morphology of NPs. The NPs were suspended in distilled water, and samples were placed onto a copper grill covered with nitrocellulose. Next, the samples were dried at room temperature and then examined using a transmission electron microscope (LEO 912AB-100; Carl Zeiss, Korea Basic Science Institute, Chuncheon) without negative staining. 2.2.5. Particle size measurement NP powder was suspended in distilled water. The average particle size of the NPs was measured using a PAR-III Laser Particle Analyzer System (Otsuka Electronics, Japan). All measurements were performed with a He-Ne laser light source (5 mW) at a 90° angle at 37 °C. In addition, the powders of NPs were suspended in 100% human blood serum to evaluate particle size stability. The particle size was measured as mentioned above. 2.2.6. Powder X-ray diffraction The crystallinity of the samples was investigated using powder X-ray diffraction (PXRD). The technique could be described briefly as follows:
2.2.8. MRI For MRI of cancer cells in vitro, an agar solution (2.5%) was prepared by heating agar powder in distilled water at 80 °C for 20 min. Cancer cells (1 × 106) were incubated with different concentrations of paclitaxel-loaded GOAS-MNPs for 6 h. Then, the cells were washed and suspended in distilled water. We thoroughly mixed 200 μL of agar solution with 800 μL of cell suspension while warming at 60 °C. An aliquot of this mixture was transferred to a 500-μL tube and then cooled to room temperature. Tubes containing NPs in gel were measured using a 4.7 T MRI scanner (Bruker Biospec, Germany). The coronal images (2 mm) were acquired at various echo times (TE) with a repetition time (TR) of 5000 ms to estimate the transversal relaxation time (T2). The signal intensities of each region of interest (ROI) in the T2 map were measured for each concentration. 2.2.9. Iron concentration An inductively coupled plasma spectrometer (ICPS-OPTIMA 7300 DV; PerkinElmer, USA) was used to determine the iron concentration in the NPs. The samples were prepared for analysis by ICPS by dissolving 10 mg of NPs in 10 mL of 35% HCl and diluting with distilled water. 2.2.10. Biodistribution and pharmacokinetic studies 2.2.10.1. Animal study. For the studies on pharmacokinetics and biodistribution, we used Sprague–Dawley rats (220–250 g) and administered Taxol® and drug-loaded NPs intravenously through the tail vein of mice (7 mg/kg). The blood, heart, liver, spleen, lung, kidney, stomach, intestine, and muscle were collected at a determined time. Then, tissue samples were homogenized in 40 g/L BSA (0.5 g tissue/3 mL of BSA). The extraction procedure was continued as described below. The pharmacokinetic parameters were calculated using the analytical bioavailability program, BA Calc 2002 (Korea). 2.2.10.2. HPLC analysis. The HPLC system (Jasco, Tokyo, Japan) used in this study consisted of a pump (PU-980), a UV–visible spectrophotometric detector (UV-975), an autosampler (Jasco, AS-950-10), and an in-line degasser (DG-980-50). Drug analysis was performed using an analytical column (150 mm × 4.6 mm, Luna 5 μm C18). A mixture of acetonitrile and water (60:40, v/v) was used as the mobile phase for the HPLC analysis of paclitaxel in plasma, while acetonitrile and water at a ratio of 45:55 (v/v) was used as the mobile phase for the analysis of paclitaxel in tissue samples. The detection wavelength was set at 230 nm. The entire solution was filtered using a 0.45-μM membrane
Table 1 Pharmacokinetic parameters of paclitaxel after administration of Taxol® and drug-loaded nanoparticles. (μg h/mL)
Injection
AUC0–8
Taxol Paclitaxel-loaded nanoparticles
5.32 ± 0.12 26.25 ± 0.81⁎
h
t1/2 (h)
CL (L/h)
V (L/kg)
5.13 ± 0.36 9.02 ± 1.15⁎
0.1877 ± 0.004 0.0177 ± 0.002⁎
1.34 ± 0.06 0.23 ± 0.01⁎
All data are presented as the mean ± standard deviation (SD, n = 3). The statistical significance of the differences was analyzed using ANOVA and then assessed using Duncan's multiple range tests. AUC0–8 h: Area under the plasma concentration-time curve from 0 to 8 h. t1/2: elimination half-life. CL: total body clearance. V: apparent volume of distribution. ⁎ Statistically significant difference compared to Taxol® (p b 0.05).
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filter (Millipore Corp., Bedford, MA) and degassed before running the HPLC analysis. The system was run at a flow rate of 1 mL/min. 2.2.10.3. Standard calibration curve. A stock solution of paclitaxel was prepared by dissolving 10 mg of paclitaxel in 50 mL of acetonitrile. Then, the solution was stored in a freezer. Furthermore, standard solutions with various concentrations of 0.5, 1, 2, 5, 10, and 20 ppm were obtained by diluting the stock solution with acetonitrile. The internal standard (IS, 140 ppm) was prepared by dissolving testosterone in acetonitrile. For the calibration curve, serial dilutions were made by adding stock solutions and IS to the plasma (or tissue samples) to obtain samples with final concentrations of 50, 100, 200, 500, 1000, and 2000 ng/mL.
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the size range of 10 to 100 nm showed the most prolonged blood circulation time. On the other hand, particles below 10 nm are rapidly removed through extravasation and renal clearance, while NPs above 200 nm are usually sequestered by the spleen and removed from cells by the phagocyte system [40]. Thus, the size of drug-loaded NPs is responsible for evading the effect of the reticuloendothelial system (RES) and enables the most effective distribution in certain tissues. Moreover, the surface charge value of − 41.89 mV for drug-loaded GOAS-MNPs implied that these NPs are stable in suspension.
2.2.10.4. Extraction procedure. The plasma or tissue samples were subjected to the same extraction procedure. Initially, the samples were stored in a freezer and allowed to thaw in a water bath at room temperature before processing. Then, the samples (100 μL) were mixed with 10 μL of the IS, and the resulting mixture was extracted with 1.5 mL of tertbutyl methyl ether and centrifuged at 1000 rpm for 3 min. The organic layer was then transferred to a clean tube and evaporated under a gentle stream of nitrogen at 40 °C in a Dry Thermo Unit DTU-1B (Taitec Corp., Tokyo, Japan). The extract was reconstituted with the mobile phase and injected into the HPLC system. Drug concentration was determined from the peak area ratios with respect to those of the IS. 2.2.11. In vivo antitumor activity The antitumor efficacy of paclitaxel-loaded NPs was evaluated in breast and colon tumor-bearing BALB/c mice. We used EMT-6 and CT26 cells as the cancer cell lines; the BALB/c mice were 7 weeks old and weighed 20–25 g. The body weight and tumor volume were determined at defined time periods to evaluate the in vivo antitumor efficacy. The tumor volume was calculated by measuring two perpendicular diameters using the formula (L × W2)/2, where L is the longest diameter and W is perpendicular to L [51]. For the treatment of breast tumor-bearing BALB/c mice, paclitaxelloaded GOAS-MNPs (2, 4, and 8 mg/kg), saline, or Taxol® (8 mg/kg) were injected via the tail vein every week (2-week treatment) when the tumor size was approximately 6 mm. Similarly, for the treatment of colon tumor-bearing BALB/c mice, paclitaxel-loaded GOAS-MNPs (2.5, 5 and, 10 mg/kg) and Taxol® (10 mg/kg) were injected via the tail vein for 12 days at intervals of 3 days when the tumor sized increased to approximately 3 mm. 2.2.12. Statistical analysis All data are presented as the mean ± standard deviation. The statistical significance of the differences was analyzed using ANOVA and then assessed using Duncan's multiple range tests. A p value b 0.05 or 0.01 was considered significant. 3. Results and discussion 3.1. Characteristics of paclitaxel-loaded GOAS-MNPs The morphology of the resulting product was examined by SEM and TEM. A typical SEM image of the paclitaxel-loaded GOAS-MNPs with a spherical shape and diameter in the nanometer range is shown in Fig. 1A. TEM was used to further and more clearly characterize the morphology of the NPs. These NPs were smaller than 100 nm (Fig. 1B). The average particle size of the paclitaxel-loaded GOAS-MNPs was 83.7 nm, which was consistent with the results from TEM imaging analysis. These drug-loaded NPs could provide more efficient exposure to the tumor through the EPR effect due to the leaky and porous vascular structure of the blood capillary system accompanied by poor lymphatic drainage [30]. Moreover, small-sized NPs size can allow drugs to accumulate in solid tumors by a filtration mechanism [39,40]. NPs within
Fig. 4. Tissue distribution of Taxol® and paclitaxel-loaded GOAS-MNPs in rats at 15 (A), 60 (B), and 480 (C) min. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
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Fig. 5. Effects of Taxol® and paclitaxel-loaded GOAS-MNPs on the growth of breast (A) and colon tumors (B) in mice. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
Furthermore, PXRD patterns were used to evaluate the crystalline structure of the original NPs, which may be affected by drug loading. Fortunately, the crystalline structure of the original NPs was not changed after loading the drug into the NPs (Fig. 1C). A clear broadening of the diffraction peaks confirmed the nanocrystalline nature of the materials [41]. The magnetic properties of the paclitaxel-loaded GOAS-MNPs were evaluated by the measurement of field-dependent magnetization (Fig. 2A). The saturation magnetization was found to be close to 40 emu/g, and the lack of hysteresis loops indicated the superparamagnetic nature of the NPs [40]. To assess the potential of paclitaxel-loaded GOAS-MNPs to act as diagnostic markers in MRI, cancer cells were incubated with
paclitaxel-loaded GOAS-MNPs, and the signal intensity was measured. The relaxation curves became steeper as the iron concentration was increased from 5 μg/mL to 40 μg/mL (Fig. 2B). Paclitaxel-loaded GOASMNPs with strong magnetization can cause microscopic magnetic field inhomogeneity, which leads to dephasing of the proton magnetic moments and thus shortening of T2 relaxation. In addition, T2-weighted MRI of the cells incubated with paclitaxel-loaded GOAS-MNPs containing various concentrations of iron oxide distinctly showed a contrast effect (Fig. 2C). The signal intensity of the T2-weighted images decreased together with an increase in iron concentration. These results clearly show that paclitaxel-loaded GOAS-MNPs can be uptaken by cells and can be used as T2-weighted MRI contrast agents in biological systems.
Fig. 6. Body weight changes of breast (A) and colon tumor-bearing-mice (B) treated with saline, Taxol®, and paclitaxel-loaded GOAS-MNPs. All data are presented as the mean ± standard deviation (SD, n = 3).
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3.2. Pharmacokinetics and biodistribution of drug-loaded NPs The plasma concentration versus time profiles of paclitaxel after intravenous injection of Taxol® and paclitaxel-loaded NPs are shown in Fig. 3. The plasma concentration of paclitaxel when administered in the form of NPs was higher than that when administered in the form of Taxol®. Paclitaxel from Taxol® injection was quickly removed from the circulation system after 15 min, while the blood clearance of paclitaxel from paclitaxel-loaded NPs was significantly delayed. Therefore, loading drugs into NPs could improve their circulation half-life. Physicochemical characteristics of NPs such as hydrophilicity, surface charge, and particle size may affect their uptake by the RES. Modification of surface properties, therefore, could improve the circulation time of NPs. The presence of GEL on the surface of the NPs avoids or retards the recognition of NPs by the RES because of its hydrophilicity [30,42–44]. In addition to hydrophilicity/hydrophobicity, neutral and negatively charged surface NPs have reduced plasma protein adsorption, which leads to a decrease in aggregation and clearance from the blood stream, while positively charged surface NPs have short blood circulation halflives [45]. Moreover, NPs with a zeta potential above (+/−) 30 mV confer stability in suspension because the aggregation of the particles is prevented [46]. The presence of a highly negative charge on the drugloaded GOAS-MNPs (−41.89 mV) could be another critical parameter for controlling the circulation time. On the other hand, it is clear that along with surface composition, the size of NPs can affect blood circulation time and therapeutic efficacy. Because the average particle size of these NPs is below 100 nm, these NPs can escape the RES and realize the most effective distribution in certain tissues [40]. The related pharmacokinetic parameters are listed in Table 1. The area under the plasma concentration curve (AUC0–8 h) of Taxol® was 5.32 ± 0.12 μg h/mL, whereas the AUC of paclitaxel-loaded NPs was 26.25 ± 0.8 μg h/mL, which was 4.93-fold higher than that of Taxol®. The elimination half-lives of paclitaxel-loaded NPs and Taxol® were 9.02 h and 5.13 h, respectively, which also showed a significant difference between the pharmacokinetics of the two formulations. Moreover, the values of clearance (CL) and apparent volume distribution (V) of the two formulations were significantly different: the CL and V values of paclitaxel-loaded NPs were 10 and 5.8 times lower than those of Taxol®, respectively. These results confirmed that our NPs could remain in circulation for a longer time than Taxol®, thus leading to a significantly higher concentration of the drug in the tumor. The biodistributions of paclitaxel-loaded NPs and Taxol® in rats were determined at different time points after intravenous injection (7 mg/kg). The concentrations of paclitaxel in tissue samples, such as the liver, spleen, kidney, heart, lung, small intestine, stomach, muscle, and plasma, as a function of time were analyzed by HPLC analysis. The distribution profiles of Taxol® and NPs loaded with paclitaxel in rats are presented in Fig. 4. After IV injection, paclitaxel was widely distributed to most tissues. Generally, the highest amount of paclitaxel was extracted from the liver, followed by the kidney, spleen, lung, and heart, after 15 min and 60 min of administration. Nevertheless, paclitaxel was eliminated after 480 min, and all the organs examined showed similar distribution profiles. The distribution of paclitaxel in the liver, spleen, and lung was regulated by its uptake by the RES. Generally, particulates (b7 μm) are taken up by the Kupffer cells in the liver, macrophages in the spleen, and alveolar macrophages in the lungs [47]. In addition to the uptake by the RES, another factor, such as the blood perfusion of the organ, may affect the accumulation of paclitaxel in some non-RES organs [47]. Paclitaxel attained peak concentration in the stomach and small intestine at 60 min instead of at 15 min, as was observed in the non-gastrointestinal tissues, which may be because of the excretion of paclitaxel into the gastrointestinal tract [47,48]. Compared to Taxol®, drug-loaded NPs showed lower distributions of paclitaxel in tissue samples. Rapid clearance of the drug from the body by the RES may limit its overall effectiveness [49]. When paclitaxel was loaded in the NPs, its uptake by the RES could be retarded. Therefore,
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the circulation half-life of paclitaxel-loaded NPs was improved and resulted in their limited transport to tissue samples [39]. 3.3. In vivo antitumor efficacy We measured the in vivo antitumor activity of Taxol® and paclitaxelloaded NPs in BALB/c mice. In addition, we measured changes in the tumor volume of breast and colon tumor-bearing mice after treatment with Taxol® and paclitaxel-loaded NPs. Saline was injected as a control formulation. Compared to treatment with saline, treatment with paclitaxel clearly inhibited the growth of breast and colon tumors (Fig. 5A, B). A statistically significant inhibition in tumor growth was observed 12 days after administration of paclitaxel-loaded GOAS-MNPs compared to that after administration of Taxol®. The accumulation of NPs in the tumor region is based on the abnormality in the endothelium of the blood vessels of the tumor, the leaky and porous vascular structure of the blood capillary system, and poor lymphatic drainage, which cause EPR [32,33,49]. Thus, our results showing the improvement in the circulatory half-life of paclitaxel-loaded NPs, which have a hydrophilic surface for reduced clearance by the RES, indicated that drug-loaded NPs extravasate into tumor tissues, accumulate, and release the therapeutic drug locally in the extracellular area [45]. Consequently, a greater amount of paclitaxel is accumulated in the tumor, which results in more effective inhibition of tumor growth by changing the local environment of the tumor [50]. The body weight of tumor-bearing mice was determined simultaneously during the study of antitumor activity. The body weight changes in tumor-bearing mice after injection of saline, Taxol®, or paclitaxelloaded NPs is shown in Fig. 6A and B. Eight days after injection, the body weights of mice treated with any of the paclitaxel formulations were almost constant and were significantly different from those of the control group. The body weight of mice treated with saline injection continuously increased because of the growth of tumor. In contrast, the tumor volumes were controlled, and the growth of the tumor was delayed by treatment with paclitaxel, which resulted in a slight increase in body weight from 8 days to 21 days. Furthermore, the rate of the increase in body weight of tumor-bearing mice depended on the dose and type of treatment. The highest dose of paclitaxel-loaded GOASMNPs or Taxol® showed the slowest increase in body weight. These results indicated that the tumor volumes of the group treated with the highest dose of paclitaxel or Taxol® was smaller than those of the other groups. In summary, loading paclitaxel into our NP system could achieve a better anticancer response than Taxol® as well as the saline injection control. Furthermore, the toxic effect of paclitaxel-loaded NPs was significantly reduced [36]. 4. Conclusions The loading of paclitaxel into GOAS-MNPs leads to improved therapeutic efficacy and lower toxicity compared to Taxol®. Furthermore, drug loading into GOAS-MNPs increased the systemic circulation time of paclitaxel. A significant inhibition of tumor growth was observed after treatment with paclitaxel. On the other hand, treatment with Taxol® caused high toxicity because of the use of Cremophor® EL. Treatment with paclitaxel-loaded NPs reduced the acute toxicity in mice. Furthermore, these NPs could be used as MRI contrast agents for the diagnosis of cancer. Thus, GOAS-MNPs could be simultaneously used as diagnostic agents in imaging and as chemotherapeutic agents with minimum side effects. Conflict of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the
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manuscript. No writing assistance was utilized in the production of this manuscript.
[22] [23] [24] [25]
Acknowledgments This research was supported by a Grant (16172MFDS542) from Ministry of Food and Drug Safety and by the Korean Health Technology R&D Project, Ministry for Health and Welfare (A092018), Republic of Korea. We would like to thank the Central Research Laboratory, Kangwon National University for the use of their PXRD, TEM and SEM instruments.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biodistribution and in vivo performance of fattigation-platform theranostic nanoparticles Thao Truong-Dinh Tran a, Phuong Ha-Lien Tran b, Hardik H. Amin c, Beom-Jin Lee c,⁎ a b c
Pharmaceutical Engineering Laboratory, Biomedical Engineering Department, International University, Vietnam National University – Ho Chi Minh City, 700000, Vietnam School of Medicine, Deakin University, Geelong, Australia Bioavailability Control Laboratory, College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea
a r t i c l e
i n f o
Article history: Received 23 March 2017 Received in revised form 25 April 2017 Accepted 7 May 2017 Available online 8 May 2017 Keywords: Paclitaxel Surface-functionalized magnetic nanoparticles Drug delivery Antitumor efficacy Acute toxicity
a b s t r a c t This study was aimed at characterizing superparamagnetic nanoparticles surface-functionalized with gelatinoleic acid (GOAS-MNPs) and loaded with paclitaxel by assessing the pharmacokinetics and biodistribution of paclitaxel in tissues and the in vivo efficacy of antitumor activity after the administration of the drug. Initially, instrumental analysis was performed to examine the particle size distribution, surface charge, and morphology of the paclitaxel-loaded GOAS-MNPs. Furthermore, we evaluated their magnetic properties and performed T2-weighted magnetic resonance imaging (MRI) on cells. We intravenously administered Taxol® and paclitaxel-loaded GOAS-MNPs and compared the pharmacokinetics, biodistribution, and antitumor efficacies of the two formulations. Determination of the pharmacokinetics and the biodistribution of paclitaxel-loaded NPs showed that this formulation increased the systemic circulation time of paclitaxel and regulated its transport to tissues. The in vivo antitumor efficacy of the paclitaxel-loaded NPs was better than that of Taxol® at the same dose. Furthermore, the paclitaxel-loaded GOAS-MNPs were found to be effective as contrast agents for enhanced MRI in cancer cells. Thus, GOAS-MNPs could be an effective diagnostic system for cancer and for the delivery of paclitaxel with better therapeutic effects and a significant reduction in toxicity. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is effective against various cancers and shows particularly promising results in patients with breast and ovarian cancers [1]. Because paclitaxel is highly hydrophobic and poorly soluble in aqueous media, it has a low therapeutic index [1,2]. Recently, increasing efforts have been made toward developing paclitaxel formulations for cancer treatment. Many studies are being performed to develop a paclitaxel formulation with reduced toxicity compared to that caused by Cremophor® EL. Various approaches, such as the formation of emulsions [3], liposomes [4], conjugates [5], nanoparticles (NPs) [6–10], lipid nanocapsules [1] and water-soluble prodrugs [11,12], have been investigated to increase drug efficacy and reduce the side effects of the Taxol® formulation. Recent advances in the field of nanotechnology have enabled the development of promising diagnostic agents for the imaging of health and disease conditions and for drug discovery and therapeutic delivery [13]. Especially, hybrid materials and inorganic materials are promising materials for theranostics [9,10,14–28]. Compared to conventional dosage ⁎ Corresponding author at: Bioavailability Control Laboratory, College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea. E-mail address: [email protected] (B.-J. Lee).
http://dx.doi.org/10.1016/j.msec.2017.05.029 0928-4931/© 2017 Elsevier B.V. All rights reserved.
forms, NPs have many advantages. In addition to protecting a drug from biodegradation, NPs can target the delivery of the drug to the site of action and reduce the side effects of chemotherapy [26,29,30]. The most promising application of NPs is their use in anticancer treatment. Generally, solid tumors show hypervascular permeability and impair lymphatic drainage, which creates enhanced permeability and retention (EPR) at the tumor site [31–33]. Thus, NPs show significant accumulation in the tumor and may be useful as sustained-release formulations for injection because of their small size. Furthermore, magnetic resonance imaging (MRI) is an appealing noninvasive approach for clinical diagnosis [34]. However, MRI may lack the sensitivity required to scan small tumors because of the low contrast between tumor tissue and normal tissues. Thus, it is very interesting to develop magnetic nanoparticles (MNPs) with dual roles as carriers for the delivery of anticancer drugs and as imaging contrast agents for biomedical applications. Iron oxide NPs can serve as contrast agents in MRI and enhance the detection of lesions within the body [35]. Therefore, MNPs could be used simultaneously to visualize tumors with MRI and to administer drug therapy. We have successfully synthesized surface-functionalized magnetic nanoparticles (GOAS-MNPs) in our previous studies [36]. These paclitaxel-loaded NPs had lower cytotoxicity but a similar anticancer effect as that of Taxol®. In this study, we characterized and evaluated the in
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vivo efficacy of this nanoparticle system as a novel drug-delivering MRI contrast agent. We determined the pharmacokinetic behavior, biodistribution, and in vivo antitumor activity of this system and compared them with those of Taxol®. These studies proved that these paclitaxel-loaded NPs had many advantages, such as better antitumor efficacy and increased systemic circulation time.
was purchased from J. T. Baker (Phillipsburg, USA). Oleic acid (OA) was supplied from Shinyo Pure Chemicals Co., Ltd. (Osaka, Japan). Triethylamine was purchased from Showa (Osaka, Japan). Agar was obtained from Difco Laboratories (Detroit, USA). Paclitaxel was supplied from Dae Woong Pharmaceutical Co., Ltd. (Seoul, Korea). 2.2. Methods
2. Materials and methods 2.1. Materials Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin mixtures and trypsin-EDTA were supplied from Gibco BRL (Carlsbad, CA, USA). EMT-6 and CT26 cell lines were purchased from American Type Culture Collection (ATCC, USA). Tetraethyl orthosilicate (TEOS), iron (II) chloride, iron (III) chloride, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), N1-(3-trimethoxysilylpropyl)diethylenetriamine (DETA), gelatin (GEL), 2,4,6-trinitrobenzenesulfonic acid (TNBS), 1-ethyl-3-(3dimethylamino-propyl)carbodiimide (EDC), Cremophor EL, 2-(Nmorpholino)ethanesulfonic acid (MES), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (MTT) were purchased from Sigma (St. Louis, MO, USA). Anhydrous dimethylformamide (DMF)
2.2.1. Preparation of surface-functionalized magnetic nanoparticles (GOASMNPs) GOAS-MNPs were developed in several primary steps according to the methods used in our previous studies [36]: (1) Iron oxide NPs, i.e., MNPs, were prepared by co-precipitation of FeCl3·6H2O and FeCl2·4H2O with ammonium hydroxide according to the method reported previously [37]. (2) Iron oxide NPs were added to 100 mL of absolute ethanol (0.5 mg/mL), 5 mL of water, 2.5 mL of ammonium hydroxide (28–30 wt%), and 1 mL TEOS. This mixture was allowed to react for 5 h. Then, silanization of the NPs was performed to functionalize the amines on the surface of the NPs [38]. We used 20 mL of 1% DETA in 1 mM acetic acid to disperse 30 mg of silica-coated iron oxide NPs. The amine-functionalized nanoparticles (AS-MNPs) were formed by a 30 min reaction at room
Fig. 1. Characterization of paclitaxel-loaded surface-functionalized magnetic nanoparticles (GOAS-MNPs) using scanning electron microscopy (SEM) (A), transmission electron microscopy (TEM) (B), and powder X-ray diffraction (PXRD) patterns (C).
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temperature. (3) Oleic acid (OA) was functionalized onto AS-MNPs by conjugation with the amino groups to form OAS-MNPs. OA was first dissolved in 10 mL of anhydrous DMF containing 1% triethylamine (DMFTEA) (19.75 mg/mL). This mixture was then activated using DCC and NHS (molar ratio, 1:1:1) for 30 min. Next, the ASMNPs were dispersed in DMF-TEA (200 mg/30 mL) to form a suspension, and the abovementioned activated OA solution was added and allowed to react for 2 h. (4) For conjugation of gelatin to the surface of OAS-MNPs, 50 mg of OAS-MNPs were dispersed in pH 7.5 solution and mixed with activated gelatin solution (25 mg gelatin + 50 mL pH 6 solution at 37 °C was reacted with EDC and NHS at a 1:2:2 M ratio for 30 min) for 2 h.
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(5) In each step above, the NPs were purified by using an external permanent magnet and washing with distilled water (or DMF, in the case of oleic acid conjugation) before drying under vacuum.
2.2.2. Preparation of paclitaxel-loaded NPs GOAS-MNPs (50 mg) were suspended in 20 mL of water at 37 °C. Paclitaxel was dissolved in ethanol at a concentration of 50 mg/mL. Then, 100 μL of the drug solution was added dropwise to the NP suspension. Then, this mixture was stirred to allow the absorption of the drug into the NPs. After 5 h, the drug-loaded NPs were separated using a magnet, washed several times with distilled water, and then dried in a vacuum dryer. Drug loading and encapsulation
Fig. 2. Magnetic properties of paclitaxel-loaded surface-functionalized magnetic nanoparticles (GOAS-MNPs): magnetization curve (A), T2-relaxation analysis curves (B), and T2-weighted magnetic resonance imaging (MRI) of cancer cells with nanoparticles containing different iron concentrations (C). Red indicates high signal intensity, and blue indicates low signal intensity.
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the samples were scanned in steps of 0.02° (diffraction angle, 2θ) at a rate of 1 s per step using a zero background sample holder through a D5005 diffractometer (Bruker, Germany) and Cu-Kα radiation at a voltage of 40 kV, 50 mA. 2.2.7. Magnetization measurements Measurements of the magnetization of NPs were performed at room temperature using a vibrating sample magnetometer (VSM, Lakeshore 700).
Fig. 3. Plasma curves of paclitaxel in Taxol® and drug-loaded MNPs. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
efficiency were determined indirectly by analyzing the supernatant and washings collected from the NPs. 2.2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was used to characterize surface morphology and particle shape. The samples were examined using a Cambridge Stereo Scan 200 (London, England) at an accelerating voltage of 15 kV. The samples were mounted onto brass stages using double-sided adhesive tape and coated with gold-palladium for 60 s under an argon atmosphere using a JEOL JPC-1100 sputter coater (JEOL, Japan). 2.2.4. Transmission electron microscopy Transmission electron microscopy (TEM) was used to investigate the morphology of NPs. The NPs were suspended in distilled water, and samples were placed onto a copper grill covered with nitrocellulose. Next, the samples were dried at room temperature and then examined using a transmission electron microscope (LEO 912AB-100; Carl Zeiss, Korea Basic Science Institute, Chuncheon) without negative staining. 2.2.5. Particle size measurement NP powder was suspended in distilled water. The average particle size of the NPs was measured using a PAR-III Laser Particle Analyzer System (Otsuka Electronics, Japan). All measurements were performed with a He-Ne laser light source (5 mW) at a 90° angle at 37 °C. In addition, the powders of NPs were suspended in 100% human blood serum to evaluate particle size stability. The particle size was measured as mentioned above. 2.2.6. Powder X-ray diffraction The crystallinity of the samples was investigated using powder X-ray diffraction (PXRD). The technique could be described briefly as follows:
2.2.8. MRI For MRI of cancer cells in vitro, an agar solution (2.5%) was prepared by heating agar powder in distilled water at 80 °C for 20 min. Cancer cells (1 × 106) were incubated with different concentrations of paclitaxel-loaded GOAS-MNPs for 6 h. Then, the cells were washed and suspended in distilled water. We thoroughly mixed 200 μL of agar solution with 800 μL of cell suspension while warming at 60 °C. An aliquot of this mixture was transferred to a 500-μL tube and then cooled to room temperature. Tubes containing NPs in gel were measured using a 4.7 T MRI scanner (Bruker Biospec, Germany). The coronal images (2 mm) were acquired at various echo times (TE) with a repetition time (TR) of 5000 ms to estimate the transversal relaxation time (T2). The signal intensities of each region of interest (ROI) in the T2 map were measured for each concentration. 2.2.9. Iron concentration An inductively coupled plasma spectrometer (ICPS-OPTIMA 7300 DV; PerkinElmer, USA) was used to determine the iron concentration in the NPs. The samples were prepared for analysis by ICPS by dissolving 10 mg of NPs in 10 mL of 35% HCl and diluting with distilled water. 2.2.10. Biodistribution and pharmacokinetic studies 2.2.10.1. Animal study. For the studies on pharmacokinetics and biodistribution, we used Sprague–Dawley rats (220–250 g) and administered Taxol® and drug-loaded NPs intravenously through the tail vein of mice (7 mg/kg). The blood, heart, liver, spleen, lung, kidney, stomach, intestine, and muscle were collected at a determined time. Then, tissue samples were homogenized in 40 g/L BSA (0.5 g tissue/3 mL of BSA). The extraction procedure was continued as described below. The pharmacokinetic parameters were calculated using the analytical bioavailability program, BA Calc 2002 (Korea). 2.2.10.2. HPLC analysis. The HPLC system (Jasco, Tokyo, Japan) used in this study consisted of a pump (PU-980), a UV–visible spectrophotometric detector (UV-975), an autosampler (Jasco, AS-950-10), and an in-line degasser (DG-980-50). Drug analysis was performed using an analytical column (150 mm × 4.6 mm, Luna 5 μm C18). A mixture of acetonitrile and water (60:40, v/v) was used as the mobile phase for the HPLC analysis of paclitaxel in plasma, while acetonitrile and water at a ratio of 45:55 (v/v) was used as the mobile phase for the analysis of paclitaxel in tissue samples. The detection wavelength was set at 230 nm. The entire solution was filtered using a 0.45-μM membrane
Table 1 Pharmacokinetic parameters of paclitaxel after administration of Taxol® and drug-loaded nanoparticles. (μg h/mL)
Injection
AUC0–8
Taxol Paclitaxel-loaded nanoparticles
5.32 ± 0.12 26.25 ± 0.81⁎
h
t1/2 (h)
CL (L/h)
V (L/kg)
5.13 ± 0.36 9.02 ± 1.15⁎
0.1877 ± 0.004 0.0177 ± 0.002⁎
1.34 ± 0.06 0.23 ± 0.01⁎
All data are presented as the mean ± standard deviation (SD, n = 3). The statistical significance of the differences was analyzed using ANOVA and then assessed using Duncan's multiple range tests. AUC0–8 h: Area under the plasma concentration-time curve from 0 to 8 h. t1/2: elimination half-life. CL: total body clearance. V: apparent volume of distribution. ⁎ Statistically significant difference compared to Taxol® (p b 0.05).
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filter (Millipore Corp., Bedford, MA) and degassed before running the HPLC analysis. The system was run at a flow rate of 1 mL/min. 2.2.10.3. Standard calibration curve. A stock solution of paclitaxel was prepared by dissolving 10 mg of paclitaxel in 50 mL of acetonitrile. Then, the solution was stored in a freezer. Furthermore, standard solutions with various concentrations of 0.5, 1, 2, 5, 10, and 20 ppm were obtained by diluting the stock solution with acetonitrile. The internal standard (IS, 140 ppm) was prepared by dissolving testosterone in acetonitrile. For the calibration curve, serial dilutions were made by adding stock solutions and IS to the plasma (or tissue samples) to obtain samples with final concentrations of 50, 100, 200, 500, 1000, and 2000 ng/mL.
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the size range of 10 to 100 nm showed the most prolonged blood circulation time. On the other hand, particles below 10 nm are rapidly removed through extravasation and renal clearance, while NPs above 200 nm are usually sequestered by the spleen and removed from cells by the phagocyte system [40]. Thus, the size of drug-loaded NPs is responsible for evading the effect of the reticuloendothelial system (RES) and enables the most effective distribution in certain tissues. Moreover, the surface charge value of − 41.89 mV for drug-loaded GOAS-MNPs implied that these NPs are stable in suspension.
2.2.10.4. Extraction procedure. The plasma or tissue samples were subjected to the same extraction procedure. Initially, the samples were stored in a freezer and allowed to thaw in a water bath at room temperature before processing. Then, the samples (100 μL) were mixed with 10 μL of the IS, and the resulting mixture was extracted with 1.5 mL of tertbutyl methyl ether and centrifuged at 1000 rpm for 3 min. The organic layer was then transferred to a clean tube and evaporated under a gentle stream of nitrogen at 40 °C in a Dry Thermo Unit DTU-1B (Taitec Corp., Tokyo, Japan). The extract was reconstituted with the mobile phase and injected into the HPLC system. Drug concentration was determined from the peak area ratios with respect to those of the IS. 2.2.11. In vivo antitumor activity The antitumor efficacy of paclitaxel-loaded NPs was evaluated in breast and colon tumor-bearing BALB/c mice. We used EMT-6 and CT26 cells as the cancer cell lines; the BALB/c mice were 7 weeks old and weighed 20–25 g. The body weight and tumor volume were determined at defined time periods to evaluate the in vivo antitumor efficacy. The tumor volume was calculated by measuring two perpendicular diameters using the formula (L × W2)/2, where L is the longest diameter and W is perpendicular to L [51]. For the treatment of breast tumor-bearing BALB/c mice, paclitaxelloaded GOAS-MNPs (2, 4, and 8 mg/kg), saline, or Taxol® (8 mg/kg) were injected via the tail vein every week (2-week treatment) when the tumor size was approximately 6 mm. Similarly, for the treatment of colon tumor-bearing BALB/c mice, paclitaxel-loaded GOAS-MNPs (2.5, 5 and, 10 mg/kg) and Taxol® (10 mg/kg) were injected via the tail vein for 12 days at intervals of 3 days when the tumor sized increased to approximately 3 mm. 2.2.12. Statistical analysis All data are presented as the mean ± standard deviation. The statistical significance of the differences was analyzed using ANOVA and then assessed using Duncan's multiple range tests. A p value b 0.05 or 0.01 was considered significant. 3. Results and discussion 3.1. Characteristics of paclitaxel-loaded GOAS-MNPs The morphology of the resulting product was examined by SEM and TEM. A typical SEM image of the paclitaxel-loaded GOAS-MNPs with a spherical shape and diameter in the nanometer range is shown in Fig. 1A. TEM was used to further and more clearly characterize the morphology of the NPs. These NPs were smaller than 100 nm (Fig. 1B). The average particle size of the paclitaxel-loaded GOAS-MNPs was 83.7 nm, which was consistent with the results from TEM imaging analysis. These drug-loaded NPs could provide more efficient exposure to the tumor through the EPR effect due to the leaky and porous vascular structure of the blood capillary system accompanied by poor lymphatic drainage [30]. Moreover, small-sized NPs size can allow drugs to accumulate in solid tumors by a filtration mechanism [39,40]. NPs within
Fig. 4. Tissue distribution of Taxol® and paclitaxel-loaded GOAS-MNPs in rats at 15 (A), 60 (B), and 480 (C) min. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
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Fig. 5. Effects of Taxol® and paclitaxel-loaded GOAS-MNPs on the growth of breast (A) and colon tumors (B) in mice. All data are presented as the mean ± standard deviation (SD, n = 3). *: statistically significant difference compared to Taxol® (p b 0.05).
Furthermore, PXRD patterns were used to evaluate the crystalline structure of the original NPs, which may be affected by drug loading. Fortunately, the crystalline structure of the original NPs was not changed after loading the drug into the NPs (Fig. 1C). A clear broadening of the diffraction peaks confirmed the nanocrystalline nature of the materials [41]. The magnetic properties of the paclitaxel-loaded GOAS-MNPs were evaluated by the measurement of field-dependent magnetization (Fig. 2A). The saturation magnetization was found to be close to 40 emu/g, and the lack of hysteresis loops indicated the superparamagnetic nature of the NPs [40]. To assess the potential of paclitaxel-loaded GOAS-MNPs to act as diagnostic markers in MRI, cancer cells were incubated with
paclitaxel-loaded GOAS-MNPs, and the signal intensity was measured. The relaxation curves became steeper as the iron concentration was increased from 5 μg/mL to 40 μg/mL (Fig. 2B). Paclitaxel-loaded GOASMNPs with strong magnetization can cause microscopic magnetic field inhomogeneity, which leads to dephasing of the proton magnetic moments and thus shortening of T2 relaxation. In addition, T2-weighted MRI of the cells incubated with paclitaxel-loaded GOAS-MNPs containing various concentrations of iron oxide distinctly showed a contrast effect (Fig. 2C). The signal intensity of the T2-weighted images decreased together with an increase in iron concentration. These results clearly show that paclitaxel-loaded GOAS-MNPs can be uptaken by cells and can be used as T2-weighted MRI contrast agents in biological systems.
Fig. 6. Body weight changes of breast (A) and colon tumor-bearing-mice (B) treated with saline, Taxol®, and paclitaxel-loaded GOAS-MNPs. All data are presented as the mean ± standard deviation (SD, n = 3).
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3.2. Pharmacokinetics and biodistribution of drug-loaded NPs The plasma concentration versus time profiles of paclitaxel after intravenous injection of Taxol® and paclitaxel-loaded NPs are shown in Fig. 3. The plasma concentration of paclitaxel when administered in the form of NPs was higher than that when administered in the form of Taxol®. Paclitaxel from Taxol® injection was quickly removed from the circulation system after 15 min, while the blood clearance of paclitaxel from paclitaxel-loaded NPs was significantly delayed. Therefore, loading drugs into NPs could improve their circulation half-life. Physicochemical characteristics of NPs such as hydrophilicity, surface charge, and particle size may affect their uptake by the RES. Modification of surface properties, therefore, could improve the circulation time of NPs. The presence of GEL on the surface of the NPs avoids or retards the recognition of NPs by the RES because of its hydrophilicity [30,42–44]. In addition to hydrophilicity/hydrophobicity, neutral and negatively charged surface NPs have reduced plasma protein adsorption, which leads to a decrease in aggregation and clearance from the blood stream, while positively charged surface NPs have short blood circulation halflives [45]. Moreover, NPs with a zeta potential above (+/−) 30 mV confer stability in suspension because the aggregation of the particles is prevented [46]. The presence of a highly negative charge on the drugloaded GOAS-MNPs (−41.89 mV) could be another critical parameter for controlling the circulation time. On the other hand, it is clear that along with surface composition, the size of NPs can affect blood circulation time and therapeutic efficacy. Because the average particle size of these NPs is below 100 nm, these NPs can escape the RES and realize the most effective distribution in certain tissues [40]. The related pharmacokinetic parameters are listed in Table 1. The area under the plasma concentration curve (AUC0–8 h) of Taxol® was 5.32 ± 0.12 μg h/mL, whereas the AUC of paclitaxel-loaded NPs was 26.25 ± 0.8 μg h/mL, which was 4.93-fold higher than that of Taxol®. The elimination half-lives of paclitaxel-loaded NPs and Taxol® were 9.02 h and 5.13 h, respectively, which also showed a significant difference between the pharmacokinetics of the two formulations. Moreover, the values of clearance (CL) and apparent volume distribution (V) of the two formulations were significantly different: the CL and V values of paclitaxel-loaded NPs were 10 and 5.8 times lower than those of Taxol®, respectively. These results confirmed that our NPs could remain in circulation for a longer time than Taxol®, thus leading to a significantly higher concentration of the drug in the tumor. The biodistributions of paclitaxel-loaded NPs and Taxol® in rats were determined at different time points after intravenous injection (7 mg/kg). The concentrations of paclitaxel in tissue samples, such as the liver, spleen, kidney, heart, lung, small intestine, stomach, muscle, and plasma, as a function of time were analyzed by HPLC analysis. The distribution profiles of Taxol® and NPs loaded with paclitaxel in rats are presented in Fig. 4. After IV injection, paclitaxel was widely distributed to most tissues. Generally, the highest amount of paclitaxel was extracted from the liver, followed by the kidney, spleen, lung, and heart, after 15 min and 60 min of administration. Nevertheless, paclitaxel was eliminated after 480 min, and all the organs examined showed similar distribution profiles. The distribution of paclitaxel in the liver, spleen, and lung was regulated by its uptake by the RES. Generally, particulates (b7 μm) are taken up by the Kupffer cells in the liver, macrophages in the spleen, and alveolar macrophages in the lungs [47]. In addition to the uptake by the RES, another factor, such as the blood perfusion of the organ, may affect the accumulation of paclitaxel in some non-RES organs [47]. Paclitaxel attained peak concentration in the stomach and small intestine at 60 min instead of at 15 min, as was observed in the non-gastrointestinal tissues, which may be because of the excretion of paclitaxel into the gastrointestinal tract [47,48]. Compared to Taxol®, drug-loaded NPs showed lower distributions of paclitaxel in tissue samples. Rapid clearance of the drug from the body by the RES may limit its overall effectiveness [49]. When paclitaxel was loaded in the NPs, its uptake by the RES could be retarded. Therefore,
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the circulation half-life of paclitaxel-loaded NPs was improved and resulted in their limited transport to tissue samples [39]. 3.3. In vivo antitumor efficacy We measured the in vivo antitumor activity of Taxol® and paclitaxelloaded NPs in BALB/c mice. In addition, we measured changes in the tumor volume of breast and colon tumor-bearing mice after treatment with Taxol® and paclitaxel-loaded NPs. Saline was injected as a control formulation. Compared to treatment with saline, treatment with paclitaxel clearly inhibited the growth of breast and colon tumors (Fig. 5A, B). A statistically significant inhibition in tumor growth was observed 12 days after administration of paclitaxel-loaded GOAS-MNPs compared to that after administration of Taxol®. The accumulation of NPs in the tumor region is based on the abnormality in the endothelium of the blood vessels of the tumor, the leaky and porous vascular structure of the blood capillary system, and poor lymphatic drainage, which cause EPR [32,33,49]. Thus, our results showing the improvement in the circulatory half-life of paclitaxel-loaded NPs, which have a hydrophilic surface for reduced clearance by the RES, indicated that drug-loaded NPs extravasate into tumor tissues, accumulate, and release the therapeutic drug locally in the extracellular area [45]. Consequently, a greater amount of paclitaxel is accumulated in the tumor, which results in more effective inhibition of tumor growth by changing the local environment of the tumor [50]. The body weight of tumor-bearing mice was determined simultaneously during the study of antitumor activity. The body weight changes in tumor-bearing mice after injection of saline, Taxol®, or paclitaxelloaded NPs is shown in Fig. 6A and B. Eight days after injection, the body weights of mice treated with any of the paclitaxel formulations were almost constant and were significantly different from those of the control group. The body weight of mice treated with saline injection continuously increased because of the growth of tumor. In contrast, the tumor volumes were controlled, and the growth of the tumor was delayed by treatment with paclitaxel, which resulted in a slight increase in body weight from 8 days to 21 days. Furthermore, the rate of the increase in body weight of tumor-bearing mice depended on the dose and type of treatment. The highest dose of paclitaxel-loaded GOASMNPs or Taxol® showed the slowest increase in body weight. These results indicated that the tumor volumes of the group treated with the highest dose of paclitaxel or Taxol® was smaller than those of the other groups. In summary, loading paclitaxel into our NP system could achieve a better anticancer response than Taxol® as well as the saline injection control. Furthermore, the toxic effect of paclitaxel-loaded NPs was significantly reduced [36]. 4. Conclusions The loading of paclitaxel into GOAS-MNPs leads to improved therapeutic efficacy and lower toxicity compared to Taxol®. Furthermore, drug loading into GOAS-MNPs increased the systemic circulation time of paclitaxel. A significant inhibition of tumor growth was observed after treatment with paclitaxel. On the other hand, treatment with Taxol® caused high toxicity because of the use of Cremophor® EL. Treatment with paclitaxel-loaded NPs reduced the acute toxicity in mice. Furthermore, these NPs could be used as MRI contrast agents for the diagnosis of cancer. Thus, GOAS-MNPs could be simultaneously used as diagnostic agents in imaging and as chemotherapeutic agents with minimum side effects. Conflict of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the
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manuscript. No writing assistance was utilized in the production of this manuscript.
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Acknowledgments This research was supported by a Grant (16172MFDS542) from Ministry of Food and Drug Safety and by the Korean Health Technology R&D Project, Ministry for Health and Welfare (A092018), Republic of Korea. We would like to thank the Central Research Laboratory, Kangwon National University for the use of their PXRD, TEM and SEM instruments.
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