The sustained delivery of temozolomide from electrospun PCL- Diol-b-PU/gold nanocompsite nanofibers to treat glioblastoma tumors
Abstract
In the present study, the PCL-Diol-b-PU/Au nanocompsite nanofibers were fabricated via electrospinning process during two different stages to load an anticancer temozolomide (TMZ) drug into the nanofibers. The first stage was the incorporation of Au nanoparticles into the nanofibers and the second stage was coating the gold nanoparticles on the surface of PCL-Diol- b-PU/Au composite nanofibers. The prepared nanofibrous formulations were characterized using FTIR, SEM and TEM analysis. Box-Behnken-design was used to investigate the influence of electrospinning parameters including solution concentration, applied voltage to tip-collector distance ratio and collector speed on the morphology and fiber diameter of PCL-Diol-b-PU/Au nanofibers. Drug loading efficiency, in vitro release profiles of TMZ from PCL-Diol-b-PU/Au and gold-coated PCL-Diol-b-PU/Au composite nanofibers as well as in vitro antitumor efficacy against U-87 MG human glioblastoma cells were carried out. The TMZ release data were well described using Korsmayer-Peppas kinetic model in which results indicated Fickian diffusion of TMZ from nanofibers. The obtained results revealed the higher efficiency of PCL-Diol-b- PU/Au@TMZ nanofibrous implants for treatment of glioblastoma tumors.
1.Introduction
The glioblastoma (GBM) is one of the most common tumors occurred within the central nervous system [1]. To treat GBM tumors, the local delivery of chemotherapeutic agents followed by radiation therapy is almost suggested [2]. The oral ingestion or intravenous injection of anticancer drugs due to their lower concentrations at around GBM tumors are limited [3]. The barriers of the central nervous system such as blood–brain barrier, the blood–cerebrospinal fluid barrier and the blood–tumor barrier prevent the diffusion of anticancer drug molecules directly into the brain tumors [1]. The use of nanofibrous polymeric implantable device is a novel alternative method for local delivery of chemotropic agents to treat brain tumors [4-6]. The higher surface area and fine pores of nanofibers prepared by electrospinning process leads to load the higher drug content in to the nanofibers compared to other drug formulations [7-10].The electrospun nanofibers with higher drug encapsulation efficiency is a useful controlled drug delivery polymeric system compared to other drug–polymer combinational implants [11]. The reduction of fiber diameter can also increase the possibility of higher loading of drug in nanofibrous scaffolds. Furthermore, the aligned nanofibers reproduce the morphological and molecular signatures of glioma migration [12-14]. Therefore, the optimization of electrospinning parameters for preparation of thinner fibers is necessary [15]. The electrospinning parameters are polymeric solution properties such as polymer weight, polymer concentration, solvent type, viscosity and conductivity; and process variables such as applied voltage, flow rate, tip – collector distance and rotating drum speed as well as ambient conditions such temperature and relative humidity [16].
In previous studies, biodegradable polymeric nanofibers of poly-(D,L-lactide-co-glycolide) (PLGA) [4, 6] poly(L-lactic acid)-poly(ethylene glycol) (PLA/PEG) [11] and poly(3- caprolactone)-PEG-poly(3-caprolactone) (PCL-PEG-PCL, PCEC) [17] nanofibers have been used to treat gliomas tumors.Polyurethanes (PU) with their excellent physical properties, higher stability and good biocompatibility are widely used in biomedical applications such as drug delivery systems and tissue engineering [18-20]. Among different grades of PUs, the controlled release of theophylline from biodegradable poly (ε-caprolactone diol) based polyurethane (PCL-Diol-b-PU) has been reported by Reddy et al.[21]. The thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)/PU nanofibers were also fabricated and their application for nifedipine release during 30 h was investigated [19]. However, there is a little study about sustained delivery of anticancer drugs from PCL-Diol-b-PU nanofibers. Furthermore, various anticancer drugs, such as doxorubicin (DOX) [10, 11, 22], paclitaxel [4-6, 11] and dichloroacetate [23] have been incorporated into the nanofibers as an anticancer formulation. There is a little study on loading of temozolomide (TMZ) molecules into the nanofiber. TMZ is an alkylating agent which due to its ability to cross the blood–brain barrier, is one of the most effective agent in the treatment of GBM [24]. Due to its short plasma half-life of 1.8 h, TMZ has been encapsulated into the polymeric micro/nano particles to obtain the controlled release of TMZ [24, 25].
Recently hybrid nanofiber delivery systems of polymer/inorganic materials have been developed for improvement of controlled drug release. Kim et al. [26] incorporated the magnetic nanoparticles into the DOX-loaded nanofibers for induction of skin cancer apoptosis.
The DOX-loaded mesoporous silica nanoparticles were successfully incorporated into the PLA nanofibers as local implantable nanofibrous scaffolds for potential postsurgical cancer treatment [27]. In another study, the efficiency of DOX-loaded electrospun nano-hydroxyapatite–PLGA composite nanofibers against epithelial cancer cell was investigated [28]. Yan et al. [29] were successfully doped the Au nanoparticles into the DOX-loaded polyvinyl alcohol/chitosan composite nanofibers for controlled release of DOX from nanofibers. Yu et al.[30] incorporated the DOX-loaded carbon nanotubes (CNTs) into the PLGA electrospun nanofibers. They revealed that the DOX molecules can be released from the PLGA/DOX@CNTs composite nanofibers in a sustained and prolonged manner. In another study, PLA, multi-walled carbon nanotubes (MWCNTs) and DOX were successfully mixed to fabricate the PLA/DOX@MWCNTs composite nanofibers via electrospinning process [31]. The prepared nanofibrous formulation showed the higher potential for localized cancer therapy.
In recent years, nanometer-scale particles such as gold nanoparticles have been successfully used in cancer therapy. The gold nanoparticles due to the small size, and low toxicity have a high potential across the blood–brain barrier [32]. Manjumeena et al. [33] incorporated the gold nanoparticles into the poly(vinyl alcohol) nanofibrous matrix. They showed that the poly(vinyl alcohol) /gold nanofibers imparted good antiproliferative activity against breast cancer cell lines and cervical cancer cell lines. In another study, Yan et al. [34] used the polyvinyl alcohol/chitosan/DOX/gold nanorods composite nanofibers for treat of ovary cancer. They promised the gold and DOX contained fibers for treating of ovary cancer and other solid malignant tumors. In the present study, Au nanoparticles were synthesized and incorporated into the poly (ε- caprolactone diol) based polyurethane (PCL-Diol-b-PU) solutions to fabricate the PCL-Diol-b-PU/Au composite nanofibers. A three-factor three-level Box-Behnken design (BBD) was used to determine the influence of electrospinning parameters including applied voltage to tip-collector distance ratio, solution concentration and collector speed on the PCL-Diol-b-PU/Au fibers diameter. Different TMZ contents were loaded into the PCL-Diol-b-PU/Au composite nanofibers. Drug loading efficiency, in vitro TMZ release from PCL-Diol-b-PU/TMZ@Au nanofibers and kinetic studies have been investigated. Then in vitro cytotoxicity of PU/TMZ@Au nanofibers against GBM cell lines was also evaluated.
2.Experimental
Hexamethylene diisocyanate (HDI), 1, 4-Butanediol (BDO) , N,N-Dimethylformamide (DMF) and Tetrahydrofuran (THF) were purchased from Merck (Merck, Germany). Polycaprolactone diol (PCL-Diol, Mw=2000 g mol-1), phosphate buffer saline (PBS, pH: 7.4), HAuCl4, sodium citrate and temozolomide were provided from Sigma-Aldrich (Aldrich, USA).The PCL-Diol-b-PU was synthesized using a method as described previously [35]. Briefly, 12 g PCL-Diol was reacted with 3.02 g HDI in a glass vial reactor at 85 °C for 3 h. Then 1.08 g BDO was added to the reactor content and the reaction was continued for further 20 min. Finally, the reactor contents were derided in a vacuum oven at 70 °C for 24 h. The synthesized PCL-Diol-b- PU were washed three times with deionized water.The Au nanoparticles were synthesized by citrate reduction of HAuCl4, as described previously [36]. Briefly, 19 mL of 4mM HAuCl4 was refluxed in THF for 5-10 min. Then 1 mL of 0.5% warm sodium citrate solution (50- 60 ˚C) was added into the solution and the reflux wascontinued for further 30 min to obtain the red solution of gold nanoparticles. The gold nanoparticles solution was filtered using a 0.2 μm Millipore syringe filter to remove any precipitate at room temperature. To investigate the influence of Au content on the morphology of nanofibers, different contents of HAuCl4 (4mM, 6mM and 8mM) were used.To fabricate the PCL-Diol-b-PU/Au nanofibers, 80, 100 and 120 mg of PCL-Diol-b-PU were dissolved in 8 mL DMF under stirring at 60 °C for 6 h. Then, 2 mL Au suspension was added to the solution and stirring was continued for further 2 h to prepare homogenous solutions of 8, 10 and 12% PCL-Diol-b-PU/Au.
The prepared solution was placed in a 5 mL plastic syringe equipped with a syringe needle (19 gauge nozzle). A high voltage (15, 20 and 25 kV) was applied between the needle and collector to produce the PCL-Diol-b-PU/Au nanofibers.For loading TMZ into the PCL-Diol-b-PU/Au nanofibers, 10, 20 and 40 mg TMZ were dispersed into the solution under stirring for further 4 h before electrospinning process.Three factor three level Box–Behnken design (BBD) was used to determine the relation between electrospinning parameters including solution concentration (8-12%), applied voltage to tip- collector distance ratio (1.2-2.0) (applied voltage: 15, 20, 25 kV and tip-collector distance:12.5 cm) and collector speed (0-2500 rpm) on the morphology and diameter of PCL-Diol-b-PU/Au nanofibers. For preparation of all nanofibrous samples, tip-collector distance of 12.5 cm and feeding rate of 1 mL h-1 were fixed. The set-up of electrospinning process was provided from Nanomeghyas Company (Iran).The gold nanoparticles were also synthesized by citrate reduction of HAuCl4 in water [37] and coated on the PCL-Diol-b-PU/Au nanofibers surface. For coating of gold nanoparticles on the surface of nanofibers, the fabricated PCL-Diol-b-PU Au nanofibers were immersed into the 200 μg mL-1 gold nanoparticles suspensions at room temperature for 12 h. Then, the gold-coated nanofibers were washed with water to remove the excess gold nanoparticles on the surface of the nanofibers.The functional groups in the PCL-Diol-b-PU and PCL-Diol-b-PU/TMZ@Au nanocomposite nanofibers were determined by using Fourier transform infrared spectrometer (FTIR, Equinox 55 FTIR spectrometer) spectra in the range of 400–4000 cm-1.
The morphology of nanofibers was characterized using scanning electron microscopy (SEM, JEOL JSM-6380) after gold coating.Solution viscosities of PCL-Diol-b-PU/TMZ@Au composites were measured using the Ubbelohde I capillary viscometer.The structure and morphology of synthesized Au nanoparticles were determined using UV- visible spectroscopy (JAS.CO V-530, Japan) and Transmission electron microscopy (TEM, Philips). The hydrodynamic diameter and size distribution of the Au nanoparticles were determined by dynamic light scattering (Malvern Instruments, Worcestershire). Stock samples ofgold nanoparticles were diluted 25 fold for measuring the hydrodynamic diameter of the nanoparticles. The quantification of the TMZ was carried out using UV-Vis spectrophotometer at λmax of 255 nm.To determine the drug loading efficiency, the prepared nanofibers containing 10, 20 and 40 mg TMZ were dessolved in DMF/THF for 4 h and the actual TMZ content was measured using UV– Vis spectrophotometer. The actual drug amount contained in the sample was back calculated from the obtained data against a predetermined calibration curve of drugs. The calibration curve of TMZ was carried out in the concentration ranging from 2 µg/mL to 15 µg/mL.The drug release from TMZ-loaded PCL-Diol-b-PU/Au nanofibers was investigated at 37 °C in pH of 7.4. Briefly, 40 mg of TMZ-loaded PCL-Diol-b-PU/Au nanofibers was transferred to a dialysis tube with molecular weight cut off 12000−14000. The dialysis tube was immersed into 20 mL of 0.05 M phosphate buffered saline (PBS, pH 7.4).
The suspensions were placed in a shaking water bath (Hidolff) under stirring for 30 days. To determine the amount of TMZ released, 2 mL released solution were taken from the dissolution medium at specified intervals (1, 3, 6, 12, 24, 48, 72, 96, 120, 168, 240, 336, 408, 504, 672 and 720 h); while an equal amountof fresh PBS was added back to the incubation media. The release experiments were conducted in triplicate, and the average data were reported.Four pharmacokinetic models including zero-order (Eq. 2), first-order (Eq. 3), Higuchi (Eq. 4)[38] and Korsmeyer-Peppas (Eq. 5) [39] were used to analyze the release mechanism of TMZfrom PCL-Diol-b-PU/Au nanofibers. The kinetic parameters were obtained by linear regression using MATLAB software.where Q0 and Q are the loaded TMZ content and TMZ released from nanofibers after time t, respectively. K0, K1, , KH,and KKP are the constant parameters of Zero-order, First-order, Higuchi and Korsmeyer-Peppas equations, respectively. Mt/M∞ is the fractional release of TMZ at time t and ‘n’ is the diffusional coefficient related to the release mechanism [39]. When n < 0.5, a Fickian diffusion controlled-release is implied; while 0.5 < n < 1.0 indicates a release non- Fickian and 1 stands for zero order (case II transport). When n value is greater than 1.0, it indicates super case II transport.U-87 MG human glioblastoma cell lines provided by the Pasteur Institute of Iran (IPI, Tehran, Iran) were cultured in RPMI with 10% fetal calf serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2. Cells were detached with 0.25% trypsin-EDTA for cytotoxicity analysis.Cell viability test was determined using a colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a tetrazole assay as previously described [10, 40]. Briefly, 3×104 cells seeded within 96-well plates. The cells were incubated at 37 °C and 5% CO2 for 24 h. After 24 h incubation, the prepared TMZ, PCL-Diol-b-PU /TMZ@Au (20 mg) nanofibers and gold- coated-PCL-Diol-b-PU /TMZ@Au (20 mg) nanofibers were prepared with 1% DMSO andtreated to the cells at the designated time points. 1% DMSO was used as negative control. Finally, the extraction solution of nanofibers was removed and replaced with 100 μL/ colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole solution and re-incubated for further 4 h. After treating the cells with Sorenson buffer (Medical Biomaterials Research Center (MBRC), Tehran, Iran), the optical density of each well was read using amicroplate reader (Multiskan MK3, Thermo Electron Corporation, USA) at a wavelength of 570 nm, and growth inhibition was calculated. 3.Results and discussion The TEM images of synthesized gold nanoparticles by citrate reduction of HAuCl4 in water (GNp-W) and THF (GNp-THF) solvents are illustrated in Fig.1. As shown, the particles size of GNp-W is smaller than that of GNp-THF. It could be attributed to the good dispersion of GNp in water solvent compared to THF solvent. The average particle size of GNp-W and GNp-THF is found to be 18 and 23 nm. Dynamic light scattering analysis was used to determine the hydrodynamic diameter and particle size distribution of GNp (Fig. 1c). As shown in Fig.1c, the narrower size distribution of GNp-W nanoparticles in the range of 10-25 nm was obtained in comparison to GNp-THF particles size distribution in the range of 9-32 nm. The average hydrodynamic diameter of GNp-W and GNp-THF were found to be 20 and 26 nm which are in good agreement with the TEM images results. The optical absorbtion spectra of both synthesized gold nanoparticles is illustrated in Fig.1d. As shown, the both gold nanoparticles were successfully synthesized with the maximum adsorption peak at 525 nm [37].By solving the statistical model (Eq. 7) and optimization of variables, the optimal uncoded values of applied voltage to tip-collector distance ratio, solution concentration and collector speed were also estimated to be 1.6 kV/cm, 9.5% and 2500 rpm, respectively. The SEM image of nanofibers in optimum condition (Fig.3) indicated the aligned thinner fibers with an average diameter of 280 nm were obtained.The influence of Au concentration on the morphology of PCL-Diol-b-PU/Au nanofibers is illustrated in Fig.4. As shown, by increasing Au content, both fiber diameter and alignment of nanofibers were gradually decreased. Degree of alignment decreased by increasing of the Au content because the increased charge improved the instability of whipping, resulting in the nonuniform splitting of the jet. Similar trend is reported for gelatin incorporated PLGA nanofibers by Meng et al. [41]. As the fiber diameter change was lower than the degree of alignment of fibers, the 4mM HAuCl4 has been selected to produce the homogenous thinner fibers (Fig. 4a).The SEM images of PCL-Diol-b-PU/Au nanofibers and PCL-Diol-b-PU/Au nanofibers containing 20 and 40 mg TMZ are illustrated in Fig.5. As shown, the morphology and diameter of nanofibers were not appreciably changed by TMZ loading into the nanofibers.The FTIR spectra of AU, TMZ, PCL-Diol-b-PU nanofibers and gold coated-PCL-Diol-b- PU/Au@TMZ nanofibers are illustrated in Fig.6. As shown, bands at 2,924 and 2,848cm−1are assigned to the asymmetrical and symmetrical –CH2 stretching, respectively. The –OH and – N=C=O absorption bands of the PCL diol and HDI are observed at 3,433 and 2,239 cm−1, respectively. The other main functional groups of PCL-Diol-b-PU nanofibers are the NH stretching at 3400-3600 cm−1, the non-hydrogen bonded –C=O stretching at 1720 cm−1, the NH bending vibration at 1553 cm−1and the C-O band at 1250 cm−1[28]. For gold-coated PCL-Diol-b- PU/TMZ nanofibers, two strong characteristic bands of TMZ at 1620 and 3400-3600 cm−1are related to C=C or C=N stretching vibration and NH stretching, respectively [36]. The absorbtion peak between 520-550 cm−1 could be attributed to the presence of gold nanoparticles on the surface of nanofibers [37]. The actual TMZ amount in the TMZ loaded-nanofibers is listed in Table 3. As shown, the actual TMZ content (based on the weight of drug used in the solutions) in the PCL-Diol-b- PU/TMZ@Au nanofibrous samples was in the range of 93.8-95.2%. The actual TMZ amount for gold-coated PCL-Diol-b-PU/ TMZ@Au nanofibers was lower than that of PCL-Diol-b- PU/TMZ@Au nanofibrous formulations. Decreases in actual TMZ content in gold-coated PCL-Diol-b-PU/ TMZ@Au nanofibers was due to releasing of TMZ from nanofibers during gold coating.The TMZ release from PCL-Diol-b-PU/TMZ 40 mg, PCL-Diol-b-PU/Au@TMZ 40 mg and gold-coated PCL-Diol-b-PU Au@TMZ 40 mg nanofibers are illustrated in Fig. 7. As shown, the burst release of 30, 32 and 24% TMZ from PCL-Diol-b-PU/TMZ, PCL-Diol-b-PU/Au@TMZ and gold-coated PCL-Diol-b-PU Au@TMZ nanofibrous formulations were observed during the first 24 hours. This behavior could be attributed to accumulation of TMZ on the surface of nanofibers. The release rate of TMZ from nanofibers during first 24 h was in order of: PCL-Diol- b-PU/Au@TMZ >PCL-Diol-b-PU/TMZ> gold-coated PCL-Diol-b-PU Au@TMZ. The lower burst release rate of TMZ from gold-coated PCL-Diol-b-PU nanofibers compared to PCL-Diol- b-PU/TMZ and PCL-Diol-b-PU/Au@TMZ nanofibrous formulations could be attributed to the migration of TMZ molecules from the gold layer on the nanofiber surfaces to PBS solution and the lower drug content in gold coated TMZ loaded nanofibers. After that, the drug diffusion from pores of nanofibers provides the possibility of further release of drug from nanofibers. As shown in Fig.7, for all three nanofibrous implants, the TMZ release rate was significantly decreased and the sustained release of TMZ from porous structures of nanofibers was obtained. This behavior could be attributed to the lower residual drug content in nanofibers and TMZ release from inner pores of nanofibrous mats. Similar trends are obtained by other researchers [40, 42].
Therefore, the sustained release of TMZ following burst release of TMZ were achieved from nanofibrous formulations.The results of fitted TMZ release data with kinetic models are summarized in Table 4. By comparing the correlation coefficients, it was found that the Korsemeyer-Peppas model (R2 > 0.988) is well described the TMZ release behavior of all three nanofibrous formulations (Fig.7). The “n” values of Korsemeyer-Peppas equation for nanofibrous samples were lower than 0.5 which indicated the Fickian diffusion of the TMZ from prepared nanofibrous formulations matrix.The results of cell viability against U-87 human glioblastoma cells are illustrated in Fig.8. As shown, the viability of U-87 cells using pure TMZ is decreased to 66, 64, 63 and 60% after 1, 3, 5 and 7 days, respectively. The higher cytotoxicity of PCL-Diol-b-PU/Au@TMZ and gold coated PCL-Diol-b-PU/Au@TMZ nanofibers are obtained compared with pure TMZ after 3, 5 and 7 days. The lower cytotoxicity of gold-coated PCL-Diol-b-PU/Au@TMZ composite nanofibers against U-87 cells compared to pure TMZ after 1 days could be attributed to lower TMZ concentration in the medium of the composite nanofibers. The more killing of U-87 cells within 7 days using PCL-Diol-b-PU/Au@TMZ and gold coated PCL-Diol-b-PU/ Au@TMZ nanofibers could be attributed to the slower release of TMZ from nanofibers. Therefore, the prepared composite nanofibers can be used as a high potential implantable device for treatment of glioblastoma tumors into the body.
4.Conclusion
PCL-Diol-b-PU and gold nanoparticles were synthesized to fabricate the PCL-Diol-b-PU/Au composite nanofibers via electrospinning process. Box–Behnken design (BBD) was used to determine the simultaneous influence of solution concentration (8-12%), applied voltage to tip- collector distance ratio (1.2-2.0) and collector speed (0-2500 rpm) on the morphology of PCL- Diol-b-PU/Au nanofibers. Based on obtained results, the optimum electrospinning parameters values were found to be 10% solution concentration, 1.6 kV/cm applied voltage to tip-collector distance ratio and 2500 rpm collector speed for fabrication of aligned thinner nanofibers. The TMZ chemotherapeutic drug has been successfully encapsulated into the PCL-Diol-b-PU/Au nanofibers. Then, the gold nanoparticles were coated on the PCL-Diol-b-PU/TMZ@Au nanofibers to enhance the antitumor activity of nanofibers against glioblastoma cells. The lower burst TMZ release during the first day and a sustained delivery manner of TMZ from nanofibers were observed. The kinetic studies indicated that the TMZ release data were best described using Korsmeyer- Peppas kinetic model. The “n “values of Korsmeyer-Peppas indicated the Fickian diffusion mechanism of TMZ release from nanofibers. The cytotoxicity of prepared nanofibers against U-87 human glioblastoma cells indicated that the gold coating on the nanofibers surface enhanced the cytotoxicity of TMZ chemical PCL-Diol-b- PU/TMZ@Au nanofibers.