Polyethylenimine-grafted multiwalled carbon nanotubes for secure noncovalent immobilization and efficient delivery of DNA.

The functionalization of carbon nanotubes (CNTs) has been carried out in various ways for numerous applications in biotechnology, including for the preparation of sensors, as scaffolds for cell growth, imaging reagents, and transporters for drug delivery. One way is to immobilize DNA onto the surface of CNTs through noncovalent interactions or covalent bonds. Covalent-bond approaches might compromise and even spoil the functions of DNA owing to chemical reactions and the difficulty in releasing DNA. Nevertheless, noncovalent approaches developed to date may only provide metastable immobilization of DNA onto the surface of CNTs. It was reported that the migration of DNA linked covalently to CNTs was retarded in gel electrophoresis but noncovalent interactions between DNA and CNTs did not completely prevent migration. Polyethylenimine (PEI) is a type of polymer with a high density of amines, thus DNA may be immobilized securely onto the surface of multiwalled carbon nanotubes (MWNTs) that have been functionalized with PEI through strong electrostatic interactions arising from these amines. Hence, we have adopted a grafting-from approach to prepare polyethylenimine-graft multiwalled carbon nanotubes (PEIg-MWNTs). DNA has been immobilized securely onto the surface of PEI-g-MWNTs as demonstrated by the total inhibition of the migration of DNA in gel electrophoresis, and PEI-g-MWNTs showed transfection efficiency for delivery of DNA that was similar to or even several times higher than that of PEI (25 K) and several orders of magnitude higher than that of naked DNA. PEI was grafted onto the surface of MWNTs by performing a cationic polymerization of aziridine in the presence of amine-functionalized MWNTs (NH2–MWNTs). NH2– MWNTs were obtained by introducing carboxylic acid groups onto the surface of MWNTs by heating at reflux in 3.0m nitric acid. The carboxylic acid groups were transformed into acyl chloride groups by treatment with thionyl chloride followed by treatment with ethylenediamine. The grafting of PEI was realized through two mechanisms, the activated monomer mechanism (AMM) or the activated chain mechanism (ACM), by which protonated aziridine monomers or the terminal iminium ion groups of propagation chains, respectively, are transferred to amines on the surface of MWNTs. (see Supporting Information.) The relative amount of PEI grafted onto the surface of MWNTs was investigated by thermogravimetric analysis (TGA) performed under nitrogen. MWNTs were thermally stable up to 600 8C (Figure 1A, curve a) whereas pure PEI degraded completely at about 500 8C (Figure 1A, curve d). At 500 8C, pristine MWNTs, NH2–MWNTs, and PEI-g-MWNTs showed negligible, about 2.3%, and 10.5% weight losses, respectively, thus PEI-g-MWNTs contained about 8.2% PEI. Grafting with PEI made PEI-g-MWNTs easy to disperse in water, and the resulting suspension was still stable after six months. However, NH2–MWNTs dispersed poorly in water and precipitation occurred within several hours (see Supporting Information). Transmission electron microscopy (TEM) provides direct evidence of grafting of PEI onto the surface of MWNTs. Figure 1B shows TEM images of PEI-g-MWNTs on a holey carbon film: individually dispersed MWNTs are separated from others. High-resolution TEM (Figure 1B, inset) indicates that PEI was grafted onto the surface of MWNTs as lumps with different sizes instead of as a uniform coating. This clumping results from the carboxylic acid groups, the ethylenediamine, and the PEI adhering preferably to the defects, which are the most active locations for chemical or physical functionalization; these defects tend to cluster at the bends along the surface of MWNTs grown by chemical vapor deposition (CVD). In Figure 1C, the H NMR spectrum of PEI-g-MWNTs in D2O is compared with that of PEI in D2O (pH 7.0). The signal at about d= 3.1 ppm for PEI-g-MWNTs is attributed to grafted PEI, but the significantly decreased mobility of the PEI chains in PEIg-MWNTs leads to broadening of the resonances. Some of the amine groups of PEI were protonated (pKa of PEI is greater than 8.0); we found that protonation or partial protonation of PEI was necessary for the formation of a stable aqueous suspension of PEI-g-MWNTs and neutralizing PEI by adjusting the pH value to 9 or higher led to precipitation of dispersed PEI-graft-MWNTs within several hours. PEI obtained by cationic polymerization of aziridine has a dendritic structure that contains primary, secondary, and tertiary amines with a molar ratio of about 1:2:1. Grafting PEI onto the surface of MWNTs should have a negligible effect on the chemistry. The migration of DNA was totally inhibited in gel electrophoresis when the weight ratio of PEI[*] Dr. Y. Liu, D. -C. Wu, Dr. W. -D. Zhang, Dr. C. -B. He Institute of Materials Research and Engineering 3 Research Link, Singapore 117602 (Singapore) Fax: (+65)6872-7528 E-mail: ye-liu@imre.a-star.edu. sg