Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early Online: 1–7 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2015.1019670
Synthesis and evaluation of single-wall carbon nanotube-paclitaxel-folic acid conjugate as an anti-cancer targeting agent Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 07/13/15 For personal use only.
Sara Tavakolifard1, Esmaeil Biazar2, Khalil Pourshamsian3 & Mohammad H. Moslemin1 1Department of Chemistry, Science and Research Branch, Islamic Azad University, Yazd, Iran, 2Department of Biomaterials
Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran, and 3Department of Chemistry, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
drug delivery vehicles, as they tend to aggregate, resulting in poor dispersion in aqueous solutions (Smart et al. 2006, Fraczek et al. 2008, Poland et al. 2008, Firme and Bandaru 2010). Moreover, some SWNTs without any functionalization have been shown to be cytotoxic (Colvin 2003, Shvedova et al. 2003, Warheit et al. 2004). To overcome this drawback, covalent and noncovalent functionalization has been used to modify SWNTs, to improve their dispersion in water and in physiological environments (Chen et al. 2001, Prato et al. 2008, Bhirde et al. 2009, Li 2010c). In recent years, significant progress has been made in the development of novel drug delivery systems. The side walls in SWNTs have been functionalized to chemically partition nanotube surfaces for attaching various species such as PEG, phospholipid (PL) and arginine-glycine-aspartic acid (RGD), to facilitate basic and practical applications for pharmacological use (Chen et al. 1998, 2001, Kam et al. 2005a, Liu et al. 2007d, Dai 2002). Such systems have been loaded with drug molecules such as doxorubicin (DOX) and paclitaxel (PTX) via p–p stacking interactions, and the release rate of DOX has even been shown to be controllable by using nanotubes with different diameters. Here, we show that much room exists for carrying out assembly of molecules on SWCNTs with covalent prefunctionalization, based on their supramolecular chemistry (Lehn 1985). SWCNTs were conjugated with various aromatic molecules, including the chemotherapeutic cancer drug PTX (which is a commonly used drug for cancer chemotherapy) with an ultrahigh loading capacity by weight, and the widely used folic acid (FA) molecule. Taxol was modified with succinic anhydride to enhance its water solubility, and FA was immobilized for targeting by conjugation with covalent bonding. These results uncover exciting opportunities for supramolecular chemistry on water-soluble SWNTs as a promising way of attaching drugs to nanotube vehicles, for applications ranging from drug delivery to chemical and biological imaging and biosensors. In this study, we describe a system which is based on PTX-succinic anhydride-modified SWCNTs, with FA as a
Abstract Single-wall carbon nanotubes (SWCNT) represent a novel nanomaterial applied in various nanotechnology fields because of their surface chemistry properties and high drug cargo capacity. In this study, SWCNT are pre-functionalized covalently with paclitaxel (PTX) – an anticancer drug, and folic acid (FA), as a targeting agent for many tumors. The samples are investigated and evaluated by different analyses such as Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), absorption spectroscopic measurements (UV-Visible), elemental analysis, and cell analyses with cancer cell line cultures. The results show good conjugation of the targeting molecule and the anticancer drug on the surface of the carbon nanotubes (CNT). This work demonstrates that the SWCNT-PTX-FA system is a potentially useful system for the targeted delivery of anticancer drugs. Keywords: cell analyses, folic acid, functionalization, paclitaxel, single-wall carbon nanotubes
Introduction Single-wall carbon nanotubes (SWCNT) are novel polyaromatic molecules with ultrahigh surface areas up to 2600 m2/g. SWNTs possess high tensile strength, are ultralight weight, and have excellent transport conductivity as well as thermal and chemical stability (Liu et al. 2009, Smart et al. 2006). Importantly, with a high aspect ratio and high surface area with many dangling bonds on the side walls, SWNTs have the potential to be modified with many different bioactive molecules such as proteins, enzymes, nucleic acids, and drugs, for chemical, biological, and medical applications (Hirsch 2002, Sun et al. 2002, Bahr and Tour 2002, Banerjee et al. 2005, Britz and Khlobystov 2006, Chen et al. 2001, Kam et al. 2005a, Bianco et al. 2005, Cherukuri et al. 2004, Liu et al. 2007a, 2007b). Nevertheless, previous studies have reported that the first generation of carbon nanotubes (CNTs) may be unsuitable as poly(ethylene glycol) (PEG)
Correspondence: Sara Tavakolifard, Department of Chemistry, Science and Research Branch, Islamic Azad University, Yazd, Iran. Tel: ⫹ 0098 9111930128. E-mail: [email protected] (Received 15 January 2015; accepted 8 February 2015)
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targeting group. These complexes were investigated using the Fourier transform infrared spectroscope (FT-IR), scanning electron microscope (SEM), and the ultraviolet–visible spectroscope (UV-Vis), and also by thermal gravimetric analysis (TGA) and elemental analysis.
Materials and methods
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Conjugation of PTX onto amide-functionalized SWNTs A 2′ hemisuccinate derivative (PTX-Suc) of PTX was prepared by the previously reported method with some modifications (Thierry et al. 2005). Briefly, PTX (200 mg, 0.2 mmol) (C47H51NO14, PTX, Xi’an Natural Field Bio-technique Co., Ltd, P. R. China) and succinic anhydride (0.2 mmol) were dissolved in CH2Cl (15 mL) at ambient temperature. After pyridine (0.001 mmol) was added, the mixture was vigorously stirred for 3 days at ambient temperature. The reaction mixture was concentrated in vacuum and was purified by silica gel column chromatography with a chloroform/ methanol mixture (97/3). The purified PTX-Suc was obtained as a white solid (Figure 1). 1HNMR (CDCL3): δ 1.02- 1.06 (m, 6 H), 1.56 (s, 3H), 1.75 (s, 3H), 1.79 (s, 1H), 2.20 (m, 1H), 2.22 (s, OAC), 2.43 (s, OAC), 2.60 (m, CH2CH2, 4H), 3. 60 (dd, CH2, 2H), 3.75 (dq, 2H), 4.48 (dd, 2H), 4.93 (dd, 2H), 5.53 (d, 1H), 5.88 (dd, 2H), 6.19 (t, 1H), 6.29 (s, 1H), 7.39 (d, NH), 7.21 (m,OH, 1H), 7.39-8.07 (m, CH-Bz, 15H), 10.19 (brs, OH, 1H). Yield: 89%, MP: 178–80 (Figure 1). Covalently functionalized SWCNTs (SWCNT, purity, ⬎ 90%, D ⫻ L, 4–6 nm ⫻ 0.7–10 μm, Aldrich chemistry, USA) were prepared by dissolving PTX-Suc (100 mg, 0.1 mmol) in dimethyl sulfoxide (DMSO, Aldrich) and activated by N-hydroxysuccinimide (0.1 mmol, 0.011 g) (C4H5NO3, NHS, Merck, UK) and 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (0.1 mmol, 0.019 g) (C8H17N3, EDC.HCl, Fluka, USA) for 4 h at room temperature to afford an NHS ester form of PTX-Suc. Subsequently, the resulting mixture was added to SWNTs, amide-functionalized (10 mg,) (Aldrich) in PBS (pH 7), and the reaction proceeded at 50°C for 24 h. Unbound excess PTX-Suc was removed by filtration and washed thoroughly with distilled water (over 10 times) and PBS, until the filtrate became free of the white color corresponding to free PTX-Suc.
Conjugation of FA onto SWNT-PTX-Suc complex A solution of FA (0.5 mmol, 0.22 g) (C19H19N7O6, FA, Merck, Germany) and NHS (0.5 m mol, 0.057 g) in 10 ml of DMSO
was mixed with a solution of EDC (0.5 m mol, 0.095 g) in 5 ml of DMSO. The activation reaction of the FA–COOH groups proceeded at 50°C with vigorous stirring for 1 h. Then, 0.5 g of SWCNT-PXL-Suc in 10 ml of DMSO and PBS (pH 7) were added to the mixture and reacted at 50°C for 18 h. Covalently functionalized SWCNT-PXL-FA was recovered after purification by distilled water (5 times) to remove unreacted FA, and the final product was obtained as a black powder after drying (Figure 2). FT-IR measurements were recorded on a Shimadzu FT-IR 4300 instrument using KBr pellets at room temperature. Also, UV-Vis (Cary-6000i, 350–1000 spectrophotometer) absorption spectroscopic measurements were recorded on a single beam UV-Vis spectrometer, using quartz cells of 1 cm path length and water as the reference solvent, at room temperature. SEM measurement was carried out on the XL30 electron microscope (Philips, Amsterdam, Netherlands) (Lecia Cambridge S 360). The samples were investigated by TGA (NETZSCH TG 209 F1 Iris) in N2 (10°C/min). Elemental analyses of carbon, hydrogen, and nitrogen were performed using a Series II 2400 (Perkin Elmer, Waltham, MA). 1H NMR spectra were recorded in DMSO using TMS as an internal standard on a Bruker DRX-500 Advance spectrometer at 500 MHz. SEM was used to study the morphology of the SWCNTs. The SEM measurement was carried out on the XL30 Philips Scanning Electron Microscope.
Cellular study The control powders (TCPS) were well cleaned and sterilized by the autoclave method. Individual samples were placed in Petri dishes using a sterilized pincer; cell culture breast cancer cell line (MCF7) was cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. They were incubated at 37°C in a humidified CO2 incubator with 5% CO2 and 95% air. The cultures were examined regularly. To evaluate the cytotoxic effect of the material on the MKN45 cell line, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) colorimetric assay was applied (10). Briefly, cells were seeded into 96-well culture plates at 10000 cells per well containing 200 μl of medium. The medium was removed 24 h after plating, and fresh medium containing a different concentration of sample was added. After incubation for 48 h, the medium was discarded O
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Figure 1. Process of modification of PTX with succinic anhydride.
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Figure 2. Process of conjugation of PTX and FA onto amide-functionalized SWNT.
and the cells were washed twice with phosphate-buffered saline and 50 μg/ml MTT solution for 4 h, and formazan crystals were dissolved by adding 100 μg of DMSO) to each well. The absorptions were measured in triplicate at 575 nm, with a background correction at 630 nm, using a microplate ELISA reader. The results were recorded as percentage absorbance relative to untreated control cells. The percentage of cell viability was calculated using the equation: [mean optical density (OD) of treated cells/mean OD of control cells] ⫻ 100.
Results UV-Vis spectrophotometric analysis was carried out after each experiment to verify the binding of PTX and FA with the functionalized SWNTs (Figure 3). According to this analysis, the absorbance peak at 240 nm was due to PTX stacked on SWNTs, which was used for analyzing the amount of molecules loaded onto the nanotubes. However, SWNT-PTX-FA dispersions exhibit a peak at 290 nm and a weak shoulder
at about 360 nm (green curve). By studying the UV-Vis absorption shifts, we successfully demonstrated the chemical conjugation of SWCNTs with FA and PTX by amide bonding. Figure 4 shows the strong carbonyl absorbance at 1719 cm⫺ 1 due to –CONH– and –COOH groups related to SWNTPTX. The peaks at around 1651 cm⫺ 1 and 3442 cm⫺ 1 apparently correspond to the –CONH– and NH2 groups of the functionalized SWNT, respectively. The bands at 3442 cm⫺ 1 and 3200 cm⫺ 1 can be related to the NH2 and NH stretches of FA or PTX conjugates of the SWCNT, respectively. All these all indicate the formation of PTX and FA conjugates in the functionalized SWNT. Figure 5 illustrates quantitative information on SWNT from the results of the TGA. In the TGA graphs of SWNT-PTX and SWNT-PTX-FA, distinct decompositions are observable. The first one (below 100°C) can be assigned to the SWNT– amide, while the second one is related to PTX as compared with the MWNT-FA thermogram. The mass loss of functionalized SWNT-PTX and SWNT-FA at 120°C is about 17.63%,
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Figure 3. (A) UV-Vis-NIR absorbance spectra of solutions of free PTX (black), SWCNT-PTX (red), SWCNT-PTX-FA (green), and FA (blue). (B) The various concentrations of PTX-loaded SWNTs (0.5 mmol, 0.3 mmol, 0.1 mmol).
and at 310°C it is about 22%. The PTX molecules react with succinic anhydride (at the –OH site) to form cleavable ester bonds and link to the functionalized SWNTs via amide bonds. This allows for decomposition of PTX from nanotubes by ester cleavage in vitro.
Elemental analysis of the functionalized SWNT determined by CHNOS analysis and atomic absorption spectrometry This method can demonstrate, qualitatively and quantitatively, the changes that occur in both the type and the percentage of elements present in pure SWCNT-CONH2 (1), SWCNT-PTX (2), SWCNT-PTX-FA (3). Apart from the carbon values, the atomic percentages of hydrogen (2.58%) and nitrogen (5.77%) of 2 (as compared with 1) indicated the functionalization of SWNTs with PTX. On the other hand,
the percentage of nitrogen increased from 5.77% to 8.38%, confirming the FA conjugate formation. Based on these data, coupled with the assumption that the atomic percentages of nitrogen and hydrogen originated from the SWNT-PTX to SWNT-PTX-FA, these results confirmed the functionalization of SWNT with PTX and FA (Table I). Other evidence for the functionalization of MWNTs comes from the SEM images. In Figure 6, SEM images of functionalized CNTs are shown. For the functionalized samples (B), a layer of uniform organic compounds is present on the surface of the nanotubes and the diameters of the samples are slightly increased (150 nm) as compared to those of the nonfunctionalized CNTs (10–20 nm). These structures are quite different from those of the initial nonfunctionalized CNT, in which the surface is relatively smooth and clean, as depicted in Figure 6.
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SWCNT-paclitaxel-folic acid conjugate as an anti-cancer targeting agent 5
Figure 4. FT-IR spectra of SWCNT (black), SWCNT-PTX (red), and SWNT-PTX-FA (green).
Cellular study Figure 7 shows the MTT assay for TCPS (control), SWCNT, SWCNT-PTX-FA, and PTX samples in different concentrations. The results showed high toxicity for the SWCNTPTX-FA sample compared to PTX (50 μl) after 48 h.
Discussion CNTs are emerging as novel nanomaterials for various biomedical applications. CNTs can be used to deliver a variety of therapeutic agents. However, the high cytotoxicity of CNTs limits their use in humans and in many biological systems. The biocompatibility and low cytotoxicity of CNTs
are attributed to their size, dose, duration, testing systems, and surface functionalization. The functionalization of CNTs improves their solubility and biocompatibility and alters their cellular interaction pathways, resulting in greatly reduced cytotoxic effects. PTX is a powerful antimitotic agent that acts against a wide range of solid tumors (You et al. 2008, Grecory and Anne 1993). Improvement in the applications of PTX in clinical therapy is limited by poor aqueous solubility, inefficient distribution, and the lack of selectivity. FA is a water-soluble vitamin that plays an important role in cell proliferation, and has also been used as a targeting molecule in micellar and liposomal systems (Nakamura et al. 2011). Tian et al. explored an efficient targeting by the CNT-based
Figure 5. TGA spectra of SWCNT (black) curve, SWCNT-PTX (red curve), and SWCNT-PTX-FA (green curve).
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Table I. Elemental analysis of the conjugated single-wall carbon nanotubes. SWCNT %C %H %N
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1 2 3
72.44 67.14 61.86
2.05 2.58 3.02
4.75 5.77 8.38
drug delivery system (DDS), with FA as the targeting ligand and quantum dots (Q dots) as fluorescent labeling probes covalently conjugated onto to the PEI-modified MWNTs via amide bonds by 1-(3-(dimethyl amino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) crosslinker. In this study, Q dots were used as fluorescent labeling probes for clearly tracking the intracellular transport and targeted delivery of functionalized multi-wall carbon nanotubes (f-MWNTs). Furthermore, PTX bonded strongly to f-MWNTs mainly through noncovalent π-π stacking interaction. The results demonstrated that improved aqueous solubility and anticancer targeting activity of the f-MWNT–PTX complexes were achieved in vitro (Tian et al. 2011). Here, we show a new type of DDS involving modified SWCNTs for controlled loading/release of the anti-cancer drug PTX. Our aim is to develop a PTX/FA drug complex with a high anticancer effect. A schematic illustration of the synthesis of the SWCNT-PTX conjugate is shown in the figure. The PTX was covalently linked to SWCNTs through a succinate linker that is known to be cleaved to release the parent drug (PTX) under physiological conditions, with a half-life of a few hours. SWCNT-PTX was synthesized in a two-step process, as seen in the figures: (i) modification of PTX with succinic acid (the cleavable linker) (Nicolaou et al. 1993), and (ii) coupling of the activated ester form of the succinic
Figure 7. MTT results of Control, SWCNT, FA-PTX modified SWNTs, and PTX in different concentrations after 48 h, with the MCF7 cell line.
acid-modified PTX to the amide groups of SWCNT under physiological conditions. These nanotube solutions were highly stable in buffer solutions, consisting of very pure, short (average length 250 nm, and relatively straight because of the short length) SWNTs by sonication and centrifugation (O’Connell et al. 2002), rather than large aggregates, as evidenced by UV-Vis-NIR absorbance .We investigated the binding of PTX/FA to SWNTs, which could be confirmed by UV-Vis spectra. As shown in Figure 3, the characteristic UV-Vis absorbance peak for PTX at 240 nm is superimposed on the characteristic SWNT-PTX absorption spectrum. It suggests that the drug was successfully loaded onto the SWNTs, which may be mainly driven by covalent stacking and hydrophobic interactions. Note that free, unbound PTX in the SWNT solution was removed thoroughly by repeated filtrations to retain only the SWNT-PTX complex. Further, the PTX-loaded SWNTs were stable in water and physiological buffers with a pH of 7.4, without any detectable release over hours.
Figure 6. SEM images of modified SWNTs (A) Nonmodified SWNTs (B) FA-conjugated PTX/SWNTs.
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SWCNT-paclitaxel-folic acid conjugate as an anti-cancer targeting agent 7 The concentration of SWNTs was measured with a molar extinction coefficient value of 7.9 ⫻ 106 M⫺ 1⋅cm⫺ 1 for an average tube length of ∼ 250 nm (the molar extinction coefficient of the solubilized SWNTs measured were determined by the absorbance at 808 nm) (Kam et al. 2005b). The concentration of PTX loaded onto SWNTs was measured by the absorbance peak at 240 nm with a molar extinction coefficient of 31.7 ⫻ 105. The quantity of PTX loaded onto the nanotubes was measured by UV-Vis-NIR spectra, for the same batches of samples. Next, we investigated the binding of FA to SWNT-PTX. After simple mixing of the amide-functionalized SWNT-PTX solution with FA at pH 7, it was kept overnight and then repeatedly filtered to remove free unbound FA in solution, which could be confirmed by UV-Vis-NIR. It can be clearly seen that the obvious absorption peak of SWNT-PTX-FA at 280 nm can be attributed to the characteristic absorption peak of FA. This suggests that the cancer-targeting ligand FA molecules had successfully anchored to the SWCNT-PTX via the amide reaction between the carboxyl group of the FA and the amide group of the SWNT segment to form the SWNT-PTX-FA conjugates. Furthermore, SWNT-PTX-FA complexes were observed to have good stability in aqueous solution. Thus, the structure of the CNT backbone could act as a suitable platform for the formation of supramolecular complexes with insoluble aromatic drug molecules.
Conclusion In this study, FA-conjugated SWNT-PTX complexes were prepared using chemical methods. According to data obtained by UV-Vis, FT-IR, and DTG spectroscopy, we demonstrated the attachment of FA and PTX to SWNTs. Microscopic analyses showed that CNTs did not undergo physical change after the conjugation process. Conjugation of the organic combination with the CNTs was confirmed using the data obtained from elemental analysis. In addition, the procedure developed in this study can be applicable for cancer therapy.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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Synthesis and evaluation of single-wall carbon nanotube-paclitaxel-folic acid conjugate as an anti-cancer targeting agent Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 07/13/15 For personal use only.
Sara Tavakolifard1, Esmaeil Biazar2, Khalil Pourshamsian3 & Mohammad H. Moslemin1 1Department of Chemistry, Science and Research Branch, Islamic Azad University, Yazd, Iran, 2Department of Biomaterials
Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran, and 3Department of Chemistry, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
drug delivery vehicles, as they tend to aggregate, resulting in poor dispersion in aqueous solutions (Smart et al. 2006, Fraczek et al. 2008, Poland et al. 2008, Firme and Bandaru 2010). Moreover, some SWNTs without any functionalization have been shown to be cytotoxic (Colvin 2003, Shvedova et al. 2003, Warheit et al. 2004). To overcome this drawback, covalent and noncovalent functionalization has been used to modify SWNTs, to improve their dispersion in water and in physiological environments (Chen et al. 2001, Prato et al. 2008, Bhirde et al. 2009, Li 2010c). In recent years, significant progress has been made in the development of novel drug delivery systems. The side walls in SWNTs have been functionalized to chemically partition nanotube surfaces for attaching various species such as PEG, phospholipid (PL) and arginine-glycine-aspartic acid (RGD), to facilitate basic and practical applications for pharmacological use (Chen et al. 1998, 2001, Kam et al. 2005a, Liu et al. 2007d, Dai 2002). Such systems have been loaded with drug molecules such as doxorubicin (DOX) and paclitaxel (PTX) via p–p stacking interactions, and the release rate of DOX has even been shown to be controllable by using nanotubes with different diameters. Here, we show that much room exists for carrying out assembly of molecules on SWCNTs with covalent prefunctionalization, based on their supramolecular chemistry (Lehn 1985). SWCNTs were conjugated with various aromatic molecules, including the chemotherapeutic cancer drug PTX (which is a commonly used drug for cancer chemotherapy) with an ultrahigh loading capacity by weight, and the widely used folic acid (FA) molecule. Taxol was modified with succinic anhydride to enhance its water solubility, and FA was immobilized for targeting by conjugation with covalent bonding. These results uncover exciting opportunities for supramolecular chemistry on water-soluble SWNTs as a promising way of attaching drugs to nanotube vehicles, for applications ranging from drug delivery to chemical and biological imaging and biosensors. In this study, we describe a system which is based on PTX-succinic anhydride-modified SWCNTs, with FA as a
Abstract Single-wall carbon nanotubes (SWCNT) represent a novel nanomaterial applied in various nanotechnology fields because of their surface chemistry properties and high drug cargo capacity. In this study, SWCNT are pre-functionalized covalently with paclitaxel (PTX) – an anticancer drug, and folic acid (FA), as a targeting agent for many tumors. The samples are investigated and evaluated by different analyses such as Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), absorption spectroscopic measurements (UV-Visible), elemental analysis, and cell analyses with cancer cell line cultures. The results show good conjugation of the targeting molecule and the anticancer drug on the surface of the carbon nanotubes (CNT). This work demonstrates that the SWCNT-PTX-FA system is a potentially useful system for the targeted delivery of anticancer drugs. Keywords: cell analyses, folic acid, functionalization, paclitaxel, single-wall carbon nanotubes
Introduction Single-wall carbon nanotubes (SWCNT) are novel polyaromatic molecules with ultrahigh surface areas up to 2600 m2/g. SWNTs possess high tensile strength, are ultralight weight, and have excellent transport conductivity as well as thermal and chemical stability (Liu et al. 2009, Smart et al. 2006). Importantly, with a high aspect ratio and high surface area with many dangling bonds on the side walls, SWNTs have the potential to be modified with many different bioactive molecules such as proteins, enzymes, nucleic acids, and drugs, for chemical, biological, and medical applications (Hirsch 2002, Sun et al. 2002, Bahr and Tour 2002, Banerjee et al. 2005, Britz and Khlobystov 2006, Chen et al. 2001, Kam et al. 2005a, Bianco et al. 2005, Cherukuri et al. 2004, Liu et al. 2007a, 2007b). Nevertheless, previous studies have reported that the first generation of carbon nanotubes (CNTs) may be unsuitable as poly(ethylene glycol) (PEG)
Correspondence: Sara Tavakolifard, Department of Chemistry, Science and Research Branch, Islamic Azad University, Yazd, Iran. Tel: ⫹ 0098 9111930128. E-mail: [email protected] (Received 15 January 2015; accepted 8 February 2015)
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targeting group. These complexes were investigated using the Fourier transform infrared spectroscope (FT-IR), scanning electron microscope (SEM), and the ultraviolet–visible spectroscope (UV-Vis), and also by thermal gravimetric analysis (TGA) and elemental analysis.
Materials and methods
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Conjugation of PTX onto amide-functionalized SWNTs A 2′ hemisuccinate derivative (PTX-Suc) of PTX was prepared by the previously reported method with some modifications (Thierry et al. 2005). Briefly, PTX (200 mg, 0.2 mmol) (C47H51NO14, PTX, Xi’an Natural Field Bio-technique Co., Ltd, P. R. China) and succinic anhydride (0.2 mmol) were dissolved in CH2Cl (15 mL) at ambient temperature. After pyridine (0.001 mmol) was added, the mixture was vigorously stirred for 3 days at ambient temperature. The reaction mixture was concentrated in vacuum and was purified by silica gel column chromatography with a chloroform/ methanol mixture (97/3). The purified PTX-Suc was obtained as a white solid (Figure 1). 1HNMR (CDCL3): δ 1.02- 1.06 (m, 6 H), 1.56 (s, 3H), 1.75 (s, 3H), 1.79 (s, 1H), 2.20 (m, 1H), 2.22 (s, OAC), 2.43 (s, OAC), 2.60 (m, CH2CH2, 4H), 3. 60 (dd, CH2, 2H), 3.75 (dq, 2H), 4.48 (dd, 2H), 4.93 (dd, 2H), 5.53 (d, 1H), 5.88 (dd, 2H), 6.19 (t, 1H), 6.29 (s, 1H), 7.39 (d, NH), 7.21 (m,OH, 1H), 7.39-8.07 (m, CH-Bz, 15H), 10.19 (brs, OH, 1H). Yield: 89%, MP: 178–80 (Figure 1). Covalently functionalized SWCNTs (SWCNT, purity, ⬎ 90%, D ⫻ L, 4–6 nm ⫻ 0.7–10 μm, Aldrich chemistry, USA) were prepared by dissolving PTX-Suc (100 mg, 0.1 mmol) in dimethyl sulfoxide (DMSO, Aldrich) and activated by N-hydroxysuccinimide (0.1 mmol, 0.011 g) (C4H5NO3, NHS, Merck, UK) and 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (0.1 mmol, 0.019 g) (C8H17N3, EDC.HCl, Fluka, USA) for 4 h at room temperature to afford an NHS ester form of PTX-Suc. Subsequently, the resulting mixture was added to SWNTs, amide-functionalized (10 mg,) (Aldrich) in PBS (pH 7), and the reaction proceeded at 50°C for 24 h. Unbound excess PTX-Suc was removed by filtration and washed thoroughly with distilled water (over 10 times) and PBS, until the filtrate became free of the white color corresponding to free PTX-Suc.
Conjugation of FA onto SWNT-PTX-Suc complex A solution of FA (0.5 mmol, 0.22 g) (C19H19N7O6, FA, Merck, Germany) and NHS (0.5 m mol, 0.057 g) in 10 ml of DMSO
was mixed with a solution of EDC (0.5 m mol, 0.095 g) in 5 ml of DMSO. The activation reaction of the FA–COOH groups proceeded at 50°C with vigorous stirring for 1 h. Then, 0.5 g of SWCNT-PXL-Suc in 10 ml of DMSO and PBS (pH 7) were added to the mixture and reacted at 50°C for 18 h. Covalently functionalized SWCNT-PXL-FA was recovered after purification by distilled water (5 times) to remove unreacted FA, and the final product was obtained as a black powder after drying (Figure 2). FT-IR measurements were recorded on a Shimadzu FT-IR 4300 instrument using KBr pellets at room temperature. Also, UV-Vis (Cary-6000i, 350–1000 spectrophotometer) absorption spectroscopic measurements were recorded on a single beam UV-Vis spectrometer, using quartz cells of 1 cm path length and water as the reference solvent, at room temperature. SEM measurement was carried out on the XL30 electron microscope (Philips, Amsterdam, Netherlands) (Lecia Cambridge S 360). The samples were investigated by TGA (NETZSCH TG 209 F1 Iris) in N2 (10°C/min). Elemental analyses of carbon, hydrogen, and nitrogen were performed using a Series II 2400 (Perkin Elmer, Waltham, MA). 1H NMR spectra were recorded in DMSO using TMS as an internal standard on a Bruker DRX-500 Advance spectrometer at 500 MHz. SEM was used to study the morphology of the SWCNTs. The SEM measurement was carried out on the XL30 Philips Scanning Electron Microscope.
Cellular study The control powders (TCPS) were well cleaned and sterilized by the autoclave method. Individual samples were placed in Petri dishes using a sterilized pincer; cell culture breast cancer cell line (MCF7) was cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. They were incubated at 37°C in a humidified CO2 incubator with 5% CO2 and 95% air. The cultures were examined regularly. To evaluate the cytotoxic effect of the material on the MKN45 cell line, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) colorimetric assay was applied (10). Briefly, cells were seeded into 96-well culture plates at 10000 cells per well containing 200 μl of medium. The medium was removed 24 h after plating, and fresh medium containing a different concentration of sample was added. After incubation for 48 h, the medium was discarded O
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Figure 1. Process of modification of PTX with succinic anhydride.
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Figure 2. Process of conjugation of PTX and FA onto amide-functionalized SWNT.
and the cells were washed twice with phosphate-buffered saline and 50 μg/ml MTT solution for 4 h, and formazan crystals were dissolved by adding 100 μg of DMSO) to each well. The absorptions were measured in triplicate at 575 nm, with a background correction at 630 nm, using a microplate ELISA reader. The results were recorded as percentage absorbance relative to untreated control cells. The percentage of cell viability was calculated using the equation: [mean optical density (OD) of treated cells/mean OD of control cells] ⫻ 100.
Results UV-Vis spectrophotometric analysis was carried out after each experiment to verify the binding of PTX and FA with the functionalized SWNTs (Figure 3). According to this analysis, the absorbance peak at 240 nm was due to PTX stacked on SWNTs, which was used for analyzing the amount of molecules loaded onto the nanotubes. However, SWNT-PTX-FA dispersions exhibit a peak at 290 nm and a weak shoulder
at about 360 nm (green curve). By studying the UV-Vis absorption shifts, we successfully demonstrated the chemical conjugation of SWCNTs with FA and PTX by amide bonding. Figure 4 shows the strong carbonyl absorbance at 1719 cm⫺ 1 due to –CONH– and –COOH groups related to SWNTPTX. The peaks at around 1651 cm⫺ 1 and 3442 cm⫺ 1 apparently correspond to the –CONH– and NH2 groups of the functionalized SWNT, respectively. The bands at 3442 cm⫺ 1 and 3200 cm⫺ 1 can be related to the NH2 and NH stretches of FA or PTX conjugates of the SWCNT, respectively. All these all indicate the formation of PTX and FA conjugates in the functionalized SWNT. Figure 5 illustrates quantitative information on SWNT from the results of the TGA. In the TGA graphs of SWNT-PTX and SWNT-PTX-FA, distinct decompositions are observable. The first one (below 100°C) can be assigned to the SWNT– amide, while the second one is related to PTX as compared with the MWNT-FA thermogram. The mass loss of functionalized SWNT-PTX and SWNT-FA at 120°C is about 17.63%,
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Figure 3. (A) UV-Vis-NIR absorbance spectra of solutions of free PTX (black), SWCNT-PTX (red), SWCNT-PTX-FA (green), and FA (blue). (B) The various concentrations of PTX-loaded SWNTs (0.5 mmol, 0.3 mmol, 0.1 mmol).
and at 310°C it is about 22%. The PTX molecules react with succinic anhydride (at the –OH site) to form cleavable ester bonds and link to the functionalized SWNTs via amide bonds. This allows for decomposition of PTX from nanotubes by ester cleavage in vitro.
Elemental analysis of the functionalized SWNT determined by CHNOS analysis and atomic absorption spectrometry This method can demonstrate, qualitatively and quantitatively, the changes that occur in both the type and the percentage of elements present in pure SWCNT-CONH2 (1), SWCNT-PTX (2), SWCNT-PTX-FA (3). Apart from the carbon values, the atomic percentages of hydrogen (2.58%) and nitrogen (5.77%) of 2 (as compared with 1) indicated the functionalization of SWNTs with PTX. On the other hand,
the percentage of nitrogen increased from 5.77% to 8.38%, confirming the FA conjugate formation. Based on these data, coupled with the assumption that the atomic percentages of nitrogen and hydrogen originated from the SWNT-PTX to SWNT-PTX-FA, these results confirmed the functionalization of SWNT with PTX and FA (Table I). Other evidence for the functionalization of MWNTs comes from the SEM images. In Figure 6, SEM images of functionalized CNTs are shown. For the functionalized samples (B), a layer of uniform organic compounds is present on the surface of the nanotubes and the diameters of the samples are slightly increased (150 nm) as compared to those of the nonfunctionalized CNTs (10–20 nm). These structures are quite different from those of the initial nonfunctionalized CNT, in which the surface is relatively smooth and clean, as depicted in Figure 6.
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SWCNT-paclitaxel-folic acid conjugate as an anti-cancer targeting agent 5
Figure 4. FT-IR spectra of SWCNT (black), SWCNT-PTX (red), and SWNT-PTX-FA (green).
Cellular study Figure 7 shows the MTT assay for TCPS (control), SWCNT, SWCNT-PTX-FA, and PTX samples in different concentrations. The results showed high toxicity for the SWCNTPTX-FA sample compared to PTX (50 μl) after 48 h.
Discussion CNTs are emerging as novel nanomaterials for various biomedical applications. CNTs can be used to deliver a variety of therapeutic agents. However, the high cytotoxicity of CNTs limits their use in humans and in many biological systems. The biocompatibility and low cytotoxicity of CNTs
are attributed to their size, dose, duration, testing systems, and surface functionalization. The functionalization of CNTs improves their solubility and biocompatibility and alters their cellular interaction pathways, resulting in greatly reduced cytotoxic effects. PTX is a powerful antimitotic agent that acts against a wide range of solid tumors (You et al. 2008, Grecory and Anne 1993). Improvement in the applications of PTX in clinical therapy is limited by poor aqueous solubility, inefficient distribution, and the lack of selectivity. FA is a water-soluble vitamin that plays an important role in cell proliferation, and has also been used as a targeting molecule in micellar and liposomal systems (Nakamura et al. 2011). Tian et al. explored an efficient targeting by the CNT-based
Figure 5. TGA spectra of SWCNT (black) curve, SWCNT-PTX (red curve), and SWCNT-PTX-FA (green curve).
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Table I. Elemental analysis of the conjugated single-wall carbon nanotubes. SWCNT %C %H %N
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1 2 3
72.44 67.14 61.86
2.05 2.58 3.02
4.75 5.77 8.38
drug delivery system (DDS), with FA as the targeting ligand and quantum dots (Q dots) as fluorescent labeling probes covalently conjugated onto to the PEI-modified MWNTs via amide bonds by 1-(3-(dimethyl amino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) crosslinker. In this study, Q dots were used as fluorescent labeling probes for clearly tracking the intracellular transport and targeted delivery of functionalized multi-wall carbon nanotubes (f-MWNTs). Furthermore, PTX bonded strongly to f-MWNTs mainly through noncovalent π-π stacking interaction. The results demonstrated that improved aqueous solubility and anticancer targeting activity of the f-MWNT–PTX complexes were achieved in vitro (Tian et al. 2011). Here, we show a new type of DDS involving modified SWCNTs for controlled loading/release of the anti-cancer drug PTX. Our aim is to develop a PTX/FA drug complex with a high anticancer effect. A schematic illustration of the synthesis of the SWCNT-PTX conjugate is shown in the figure. The PTX was covalently linked to SWCNTs through a succinate linker that is known to be cleaved to release the parent drug (PTX) under physiological conditions, with a half-life of a few hours. SWCNT-PTX was synthesized in a two-step process, as seen in the figures: (i) modification of PTX with succinic acid (the cleavable linker) (Nicolaou et al. 1993), and (ii) coupling of the activated ester form of the succinic
Figure 7. MTT results of Control, SWCNT, FA-PTX modified SWNTs, and PTX in different concentrations after 48 h, with the MCF7 cell line.
acid-modified PTX to the amide groups of SWCNT under physiological conditions. These nanotube solutions were highly stable in buffer solutions, consisting of very pure, short (average length 250 nm, and relatively straight because of the short length) SWNTs by sonication and centrifugation (O’Connell et al. 2002), rather than large aggregates, as evidenced by UV-Vis-NIR absorbance .We investigated the binding of PTX/FA to SWNTs, which could be confirmed by UV-Vis spectra. As shown in Figure 3, the characteristic UV-Vis absorbance peak for PTX at 240 nm is superimposed on the characteristic SWNT-PTX absorption spectrum. It suggests that the drug was successfully loaded onto the SWNTs, which may be mainly driven by covalent stacking and hydrophobic interactions. Note that free, unbound PTX in the SWNT solution was removed thoroughly by repeated filtrations to retain only the SWNT-PTX complex. Further, the PTX-loaded SWNTs were stable in water and physiological buffers with a pH of 7.4, without any detectable release over hours.
Figure 6. SEM images of modified SWNTs (A) Nonmodified SWNTs (B) FA-conjugated PTX/SWNTs.
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SWCNT-paclitaxel-folic acid conjugate as an anti-cancer targeting agent 7 The concentration of SWNTs was measured with a molar extinction coefficient value of 7.9 ⫻ 106 M⫺ 1⋅cm⫺ 1 for an average tube length of ∼ 250 nm (the molar extinction coefficient of the solubilized SWNTs measured were determined by the absorbance at 808 nm) (Kam et al. 2005b). The concentration of PTX loaded onto SWNTs was measured by the absorbance peak at 240 nm with a molar extinction coefficient of 31.7 ⫻ 105. The quantity of PTX loaded onto the nanotubes was measured by UV-Vis-NIR spectra, for the same batches of samples. Next, we investigated the binding of FA to SWNT-PTX. After simple mixing of the amide-functionalized SWNT-PTX solution with FA at pH 7, it was kept overnight and then repeatedly filtered to remove free unbound FA in solution, which could be confirmed by UV-Vis-NIR. It can be clearly seen that the obvious absorption peak of SWNT-PTX-FA at 280 nm can be attributed to the characteristic absorption peak of FA. This suggests that the cancer-targeting ligand FA molecules had successfully anchored to the SWCNT-PTX via the amide reaction between the carboxyl group of the FA and the amide group of the SWNT segment to form the SWNT-PTX-FA conjugates. Furthermore, SWNT-PTX-FA complexes were observed to have good stability in aqueous solution. Thus, the structure of the CNT backbone could act as a suitable platform for the formation of supramolecular complexes with insoluble aromatic drug molecules.
Conclusion In this study, FA-conjugated SWNT-PTX complexes were prepared using chemical methods. According to data obtained by UV-Vis, FT-IR, and DTG spectroscopy, we demonstrated the attachment of FA and PTX to SWNTs. Microscopic analyses showed that CNTs did not undergo physical change after the conjugation process. Conjugation of the organic combination with the CNTs was confirmed using the data obtained from elemental analysis. In addition, the procedure developed in this study can be applicable for cancer therapy.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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