The covalent bioconjugate of multiwalled carbon nanotube and amino‐modified linearized plasmid DNA for gene delivery

Abstract

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

Recent Advances in Nanomaterials for Gene Delivery—A Review

“…Yet, when functionalized with CS-FA NPs, the transfection efficiency was increased, while the cytotoxicity decreased [63]. Geyik et al [64] delivered a linearized plasmid via covalently functionalized carboxylated MWNTs (fCNT) (Figure 3a,b). Amino groups were introduced into the plasmid DNA, resulting in amino-modified plasmid DNA (mpDNA).…”

“…Amino groups were introduced into the plasmid DNA, resulting in amino-modified plasmid DNA (mpDNA). The activated fCNTs were complexed with mpDNA to form an active bioconjugate that could be used to transform E. coli with greater transformation efficiency, and offer a potential alternative to electroporation or heat shock-induced transformation [64]. Jain et al [65] designed a different bioconjugate to deliver a plasmid to MCF-7 or HeLa cells.…”

“…Scale bar equals 0.2 μm in (a); 50 nm in (b). Figures reproduced with permission from [64]. Copyright John Wiley & Sons, 2013.…”

Applications of Functionalized Carbon Nanotubes for the Therapy and Diagnosis of Cancer

“…Similarly, Geyik et al [28] transfected plasmid DNA using carboxylated MWCNTs. In their study, after the amino-modified linear plasmid DNA and the carboxylated MWCNTs were covalently bonded, the resulting complexes were successfully transformed into competent Escherichia coli cells in the absence of a heat-shock step at 42 °C, which is usually required for regular transformation processes.…”

Evaluation of chemical modification effects on DNA plasmid transfection efficiency of single-walled carbon nanotube–succinate– polyethylenimine conjugates as non-viral gene carriers

“…The ability of SWNTs to enter cells has led to a variety of studies aimed at creating new vehicles for drug, 8,9 gene [10][11][12][13][14][15] and protein 16 delivery. Unaltered DNA does not form direct π-π stacking or hydrophobic interactions with SWNTs.…”