Functionalization of Carboxylated Multiwall Nanotubes with Dapsone Derivatives and Study of their Antibacterial Activities against E . coli and S . aureus

Bacteria are becoming resistant to the antibiotics prescribed and therefore new treatments are essential to be developed. Multiwall carbon nanotubes (MWNTs) have interesting antibacterial activities and offering a promising new treatment preventing bacteria from becoming resistant. With this aim, we report for the first time three novel modified carboxylated multiwall carbon nanotubes consisting of MWNT-Dapsone, MWNT-Dapsone-imine and MWNT-Dapsone-imine. Copper complex. The functionalized carboxylated multiwall nanotubes were then characterized by FT-IR, Raman, TEM. Moreover, the antibacterial activity of the MWNT-COOH (A), MWNTdapsone (B), MWNT-Dapsone-imine (C) MWNT-Dapsoneimine.Copper complex (D) , Dapsone (E(, Dapsone-imine (F), has been investigated against gram-negative Escherichia coli and grampositive Staphylococcus aureus. These results show that compund F exhibited significant antibacterial activity and have a potential to be used as antibacterial agent.


INTRODUCTION
Antimicrobial resistance is fast becoming a major problem with rapid increases in multidrugresistant bacteria (Saha et al., 2009).In general, bacteria have the genetic ability to send and acquire resistance to drugs, which are used as pharmacologic agents (Faúndez et al., 2004).The microbial resistance represent a global concern and the outlook for the use of antimicrobial drugs in the future is still uncertain (Stanila et al., 2011;Chaudhary et al., 2010).
Recently, carbon nanotubes (CNTs) have attracted increasing attention in biomedical fields due to their unique structure and properties, including high aspect ratios, large surface areas, nanosized stability and rich surface chemical functionalities (Wu et al., 2011;Azizian et al., 2013).With these unique structures and properties, CNTs have been developed as promising nanoplatforms to immobilize biological or therapeutic molecules, such as proteins, antibodies, siRNA or drugs on their surface, and especially these functionalized CNTs are capable of crossing biological barriers independently of the cell type, which makes them suitable candidates for drug delivery systems.(Tian et al., 2011;Hu et al., 2009;Holzinger., 2001).The chemical modification of carbon nanotubes has received significant attention in recent decades (Chen et al., 1998;Hamon et al., 1999).Chemical functionalization is a common technique to increase dispersion stability and biocompatibility of CNTs (Georgakilas et al., 2002;Sun et al., 2002).
CNT have been proposed as multipurpose innovative transporters for drug delivery since they can be covalently or non-covalently attached to drug molecules and carry them throughout the body in a biocompatible way (Karchemski et al., 2012;Entezari et al., 2013).Modified carbon nanotubes have been widely studied for their antibacterial, antifungal and potential cytotoxic chemotherapeutic agents.Many factors may influence the antibacterial activity of carbon nano materials, including: electronic structure, size and surface chemical properties, as well as the interacting conditions between carbon nanomaterials and bacterial cells.(Liu et al., 2012).More recently, transition metal complexes have attracted attentions of inorganic, metallo-organic as well as bio-inorganic chemists because of their structural diversity, antibacterial activity and enormous number of biological applications (Safari et al., 2013;Yousefi et al., 2012).The ability of metal to combine with ligands and then release ligand in specific process make them ideal candidate for use in biological system.It is well known that synthetic copper(II) complexes have been reported to act as potential anticancer and antibacterial agents (Tella and Obaleye, 2009 ).The pharmacological activities of these metal complexes depend on the metal ion, organic ligands and the structure of the compounds (Yousefi et al., 2014).Dapsone ((4, 4'-diaminodiphenylsulphone), a sulphone analog, is the main antileprosy drug because of its inherent level of bactericidal activity, easy application and minimal side effects (Makarov et al ., 2006;Wadher et al., 2009).As an extension of this research field, we aimed to attach the new pharmacologic agents to the surface of carboxylated multiwall nanotubes, in order to take advantage of the MWNT's outstanding biological properties and to screen the final products for their antibacterial activity.
In spectrum (a), the band at around 1568 cm -1 corresponds to the stretching mode of the C=C double bond that forms the framework of the carbon nanotube sidewall .The peaks at 1705 and 2450 cm -1 to 3400 cm -1 apparently corresponds to the stretching modes of the carboxylic acid groups.The two bands at around 2850 cm -1 which are seen in all spectrums are attributed to the CH stretching of MWNT-COOH defects.In spectrum (b), the three bands at 3143 cm -1 , 3304 cm -1 and 3381 cm -1   In spectrum (d), the broad band at around 1588 cm -1 corresponds to the stretching mode of the (C =O).The peak at 1625 cm -1 presents in compound (C) was shifted to lower frequency of 1463 cm -1 in compound (D), respectively, thus indicating coordination of the metal ion to N atom.Furthermore, the nature of the metal-ligand bonding is confirmed by the newly formed bands at ~ 543 cm -1 in the spectra of this compound, which is tentatively assigned to Cu-O vibration.

Raman Spectroscopy
Raman spectra offer useful information concerning the slightly structural changes of MWNTs, especially the changes owing to significant sidewall modification.As can be seen in Figure 2, the characteristic peaks of MWNT tangential modes, namely the D band at around 1320 cm -1 and the G band at around 1590 cm -1 slightly changed.We observed an increase in the ratio of intensities R = ID/IG in modified nanotubes.This indicates an increased disorder of the graphitic structure of the modified nanotubes, which shows that the nanotubes were covalently modified ( Dresselhaus et al., 2005).

Biological studies Antibacterial activity
The antibacterial activity of MWNT-COOH (A) as well as corresponding functionalized MWNTs (B-D) and compound E and F was performed against one Gram positive (Staphylococcus aureus) and another Gram negative (Escherichia coli) bacteria, and the results are summarized in Table 1.The results indicates that the modified MWNTs show higher activity than MWNT-COOH against these two bacteria (Fig 4).These data also demonstrate that MWNT-COOH has no activity on the growth of S. aureus bacteria.Compound E possessed a significant inhibitory effect against all the tested bacteria.The increase of the zone of inhibition for compound C when compared with corresponding ligands ( compound F) is an indication that the modified MWNTs is able to decrease the population of Staphylococcus aureus bacteria.Moreover, Compounds (D) present higher activity than other functionalized MWNTs against E. coli.Therefore, it should be noted that presence of metal ion in the structure of compound D had a great influence on As these results are preliminary, further study on the antibacterial activity of these complexes is highly recommended.

Material and methods
All chemicals and reagents were purchased from Merck and used without further purification.MWNT-COOH (95 % purity, 20-30 nm; Netrino Co. Ltd) were purchased and used as received.The FT-IR spectra were recorded on a Nexus 870 FT-IR spectrometer using KBr pellets (Thermo Nicolet, Madison, WI).FT-Raman spectra were recorded on 960 ES spectrometer (Thermo Nicolet).TEM measurement was carried out on the LEO 912 AB electron microscope.

Preparation of MWNT-COCl
60 mg of the MWNT-COOH were sonicated in 90 ml of DMF for 40 min to give a suspension.Oxaly-chloride (2.5 ml) was added drop-wise to the MWNT suspension at 0°C under N 2 .The mixture was stirred at 0°C for 2 h and then at room temperature for another 2 h.Finally, the temperature was raised to 70 °C and the mixture was stirred overnight to remove excess oxalyl chloride (Figure 5).After cooling to room temperature, the mixture was filtered through 0.2 µm pore size polytetrafluoroethylene membrane.The filtrate was washed with EtOH (3*100 ml), and then dried in an oven for 4 h at 60 °C.

Figure 3
presents TEM images of the MWNT-COOH (A) and the modified MWNTs (B-D).The MWNT-COOH show almost smooth surface (a) while increased roughness of the functionalized CNT surfaces evident in TEM images of MWNT-Compounds.

Table 1 . Antibacterial screening data of Compound A-E against the tested bacteria (inhibition zone in mm)
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