Imidazoles and their derivatives are compounds with chemotherapeutic applications. In this study, we investigated the chemical functionalization of carboxylated multiwalled carbon nanotubes (MWNT–COOH) by 1,2-phenylendiamine. Multiwalled nanotube (MWNT)–benzimidazole was obtained by an MWNT–amide reaction with POCl3 after 72 hours, which was confirmed by Fourier transform infrared, scanning electron microscopy, thermal gravimetric analysis, and elemental analysis. These functionalizations were chosen due to -NH2 and NHCO active sites in MWNT–amide for future application. Toxicity assays with fibroblast cells and MTT test for measurement of viable cell numbers were also performed. Cellular results did not show any toxicity change in modified samples from that of the reference samples.
Incorporation of imidazole and benzimidazole nuclei is an important synthetic strategy in drug discovery.
Compounds such as imidazole are also well known antitumor agents.
MWNT–COOH 60 mg (20–30 nm; Netvino Co. Ltd) were sonicated in 90 mL of N,N-dimethyl formamide (DMF) for 45 minutes to give a homogeneous suspension. Oxalyl chloride (2.5 mL) was added dropwise to the MWNT suspension at 0°C under nitrogen. The mixture was stirred at 0°C for two hours and followed at room temperature for the same duration. Finally, the temperature was increased to 70°C and the mixture was stirred overnight to remove excess oxalyl chloride. 1,2-phenylendiamine 100 mg dissolved in DMF was added to the MWNT suspension and the mixture stirred at 95°C for 72 hours. After cooling to room temperature, the mixture was filtered and washed thoroughly with DMF, ethyl alcohol, and tetrahydrofuran. Subsequently, the black solid was vacuum-dried at room temperature for five hours.
MWNT–amide 30 mg was mixed with 10 mL POCl3 and then stirred at 80°C for 72 hours. After cooling to room temperature, the reaction mixture was separated by centrifugation and washed thoroughly with tetrahydrofuran. Thereafter, the solid obtained was vacuum-dried for four hours.
All reagents and solvents (oxalyl chloride, 1,2-phenylendiamine, phosphoryl trichloride, and DMF) were obtained from Merck Chemical Inc. (Darmstadt, Germany), and MWCNT–COOH (95% purity, 20–30 nm; Netvino Co. Ltd) were purchased and used as received. The FT-IR spectrum was recorded using KBr tablets on a Nexus 870 FT-IR spectrometer (Thermo Nicolet, Madison, WI). SEM was used to study the morphology of the WCNTs. SEM measurement was carried out on the XL30 electron microscope (Philips, Amsterdam, Netherlands). Elemental analyses of carbon, hydrogen, and nitrogen were performed using a Series Π 2400 (Perkin Elmer, Waltham, MA). The samples were investigated by TGA (DuPont Instrument 951; DuPont, Wilmington, DE) in air (10°C/min).
A fibroblast cell suspension (L929) from mouse tails was prepared according to ISO10993standards. The powders to control (TCPS) were well cleaned and sterilized by the autoclave method. Individual samples were placed in Petri dishes using a sterilized pincer; 3 cc of the cell suspension were removed by pipette and poured into the control and experimental samples. Thereafter, all of the samples were separately placed in a Memmert incubator at 37°C for 24 and 48 hours. The samples in the polystyrene Petri dish were removed from the incubator after 24 and 48 hours and studied using a Eclipse TS-100 photonic microscope (100 ×; Nikon, Tokyo, Japan). Cell proliferation was determined by MTT assay for viable cell numbers. The MTT tetrazolium compound is reduced by living cells into a colored formazan product that is soluble in tissue culture medium. The quantity of formazan product is directly proportional to the number of viable cells in the culture. The assays were performed by adding 1 mL of MTT solution (Sigma, St. Louis, MO) and 9 mL fresh medium to each well after aspirating the spent medium and incubating at 37°C for four hours with protection from light. Colorimetric measurement of formazan dye was performed at a wavelength of 570 nm using a Rayto microplate reader.
Elemental analyses of the modified MWNT 1–3 are shown in
Raman spectroscopy is a powerful tool used to provide structural information about MWNT–COOH before and after functionalization. As shown in
More direct evidence for the functionalization of MWNTs comes from the SEM images.
In summary, we have introduced benzimidazole groups onto the surface of nanotubes via reaction of MWNT–amide with POCl3. Functionalization was demonstrated by their SEM images, as well as FT-IR, elemental analysis, and TGA. The results show successful functional groups and no change in toxicity in functional samples compared with the primary sample, with half of the cell growth on modified samples. In subsequent studies, antitumoral investigations of modified samples will be evaluated.
Funding for this research was provided by the Research Vice Presidency of Science and Research Branch, Islamic Azad University and Iranian Nanotechnology Initiative (Government of Iran).
The authors report no conflicts of interest in this work.
Synthesis route of the modified carboxylated multiwalled carbon nanotubes.
Fourier transform spectra (after baseline correction) of functionalized carbon nanotubes.
Raman spectra of carboxylated multiwalled carbon nanotubes and benzimidazole multiwalled carbon nanotubes.
Scanning electron microscopy images of
Thermal gravimetric analysis curves of modified multiwalled carbon nanotubes in air (10°C/min).
Cell growth on the samples. Control
Elemental analysis of the modified multiwalled carbon nanotubes (MWNT)
| MWNT | %C | %H | %N |
|---|---|---|---|
| 1 | 96.4 | 0.04 | 0 |
| 2 | 89.7 | 1.31 | 2.09 |
| 3 | 85.2 | 0.96 | 1.47 |
MTT analysis of the samples
| Sample | λ(nm) | Vialibility% |
|---|---|---|
| Control | 630 | 100 |
| MWNT–Amide | 317 | 50 |
| MWNT–Benzimidazole | 332 | 53 |
| MWNT–COOH | 347 | 55 |