Elsevier

Carbon

Volume 50, Issue 8, July 2012, Pages 2691-2697
Carbon

Monitoring the functionalization of single-walled carbon nanotubes with chitosan and folic acid by two-dimensional diffusion-ordered NMR spectroscopy

https://doi.org/10.1016/j.carbon.2012.02.010Get rights and content

Abstract

A conjugate between single-walled carbon nanotubes, chitosan and folic acid has been prepared. It was characterized by diffusion ordered two-dimensional hydrogen-1 nuclear magnetic resonance and hydrogen-1 nuclear magnetic resonance spectroscopy which revealed the presence of a conjugate that was generated by the linkage between the carboxyl moiety of the folic acid and the amino group of the chitosan, which in turn was non-covalently bound to the single-walled carbon nanotubes. The obtained diffusion coefficient values demonstrated that free folic acid diffused more rapidly than the folic acid conjugated to single-walled carbon nanotubes–chitosan. The values of the proton signal of hydrogen-1 nuclear magnetic resonance spectroscopy and two-dimensional hydrogen-1 nuclear magnetic resonance spectroscopy further confirmed that the folic acid was conjugated to the chitosan, wrapping the single-walled carbon nanotubes.

Introduction

Over the past decade, the interest for using carbon nanotubes (CNTs) functionalized with different types of biomolecules has increased [1], [2], [3]. The nanometer size and unique physical properties of CNTs make them attractive for anticancer drug delivery [4], [5], [6], [7], [8], molecular transport [9], [10], [11], and new therapeutic mechanisms [12], [13]. CNTs have been activated to target various cancer cells using folic acid (FA). FA is an attractive ligand that is useful for targeting cell membranes and enhancing CNTs endocytosis by the folate receptor [14], [15]. FA receptors can be direct targets for drug delivery [16], which explains the diversity of strategies used for folate conjugation.

Conventionally, CNTs–FA has been characterized by UV–Vis (ultraviolet–visible spectroscopy), NIR spectroscopy (near-infrared) [17], AFM (atomic force microscopy) and TEM (transmission electronic microscopy) [9] but not with 2D NMR (two-dimensional nuclear magnetic resonance) experiments. The 2D NMR techniques have become popular since they efficiently map out 3D interactions within, or sometimes between, molecules [18]. The diffusion ordered spectroscopy (DOSY) technique is a non-invasive powerful technology that has been used for the analysis of a large variety of mixtures [19], as well as for the characterization of aggregates of varying sizes and hence different diffusion coefficients (DC) [20].

The DOSY technique has been referred to as “NMR chromatography” for its ability to “separate” the components of a complex mixture. In a recent application, Marega et al. [21] used 1H-2D DOSY spectroscopy to monitor the functionalization and purification of a carbon nanotube–polyethylene glycol (CNT–PEG) conjugate. There have been numerous publications on the characterization of various bioconjugates using 2D NMR techniques [22], [23]. However, studies related to the characterization of CNT–FA conjugates using 2D NMR spectroscopy have yet to be published.

The aim of this study was to characterize a conjugate formed by single-walled carbon nanotubes (SWCNTs), chitosan and FA by means of the DOSY 2D-NMR technique 1H NMR spectroscopy. The values obtained for the diffusion coefficient confirmed the conjugation of FA to SWCNTs wrapped by the chitosan. The synthesis and utilization of these conjugate systems opens new possibilities for the treatment of infectious diseases and cancer cells [24].

Section snippets

Materials

SWCNTs with diameters ranging from 2 to 5 nm and lengths between 500 and 2000 nm were purchased from Unydim, California. Chitosan with a molecular weight of 5000 Da, folic acid (FA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Sigma–Aldrich Corp. These and all other chemicals used in this work were of analytical grade.

Solubilization of single-walled carbon nanotubes

Five milligrams of SWCNTs were sonicated for 1 h in 5 mL of a 30% aqueous solution of chitosan. The solution was centrifuged at 2800 rpm for

Results and discussion

The conjugate was prepared by a reaction of FA with SWCNT–chitosan in the presence of EDC (Fig. 3). The EDC is the most popular carbodiimide for conjugating biological substances containing carboxylates and amines [23] and it was used as a crosslinking agent to mediate the formation of an amide between the carboxyl group of the folic acid and the amine group of the chitosan. Hydrolysis by water of the intermediate (EDC–FA) is the major competing reaction cleaving off the activated ester

Conclusions

According to the present study, a two-dimensional diffusion-ordered NMR spectroscopy with presaturation of the solvent (ZGPR) could be successfully employed to monitor the conjugation of folic acid to SWCNTs–chitosan. The obtained diffusion coefficient values revealed that free folic acid diffused more rapidly than its counterpart conjugated to SWCNT, thus proving the efficiency of the synthesis method when it comes to attaching the folic acid molecules around the SWCNT. This is an important

Acknowledgments

The Colombian Administrative Department of Science, Technology and Innovation, COLCIENCIAS (Project 110245921468) and the Danish Agency for Science Technology and Innovation (FSS 09-066053) are gratefully acknowledged for financial support. Doctor Casper H. Clausen who help with the AFM image.

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