Binding and Condensation of Plasmid DNA onto Functionalized Carbon Nanotubes:  Toward the Construction of Nanotube-Based Gene Delivery Vectors

Abstract

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.

Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to haptens

“…[39][40][41][42][43] They are capable of crossing membranes to penetrate 80 into different types of cells without causing cell death, so both the cell functionality and the activity of the introduced molecules are preserved. [44][45][46] Based on this finding, CNTs have been successfully employed as nanovehicles for drug delivery [43,47,48] and for the transport of biomolecules, such as proteins, [49] peptides, [50] and DNA[42, 51,52], into cells. Additionally, based on their structural and chemical properties, CNTs can be envisioned as excellent carrier 85 candidates in immunization protocols.…”

Carbon Nanotube‐based Vectors for Delivering Immunotherapeutics and Drugs

“…CNTs have very recently been explored for the first time as novel materials for the complexation and delivery of gene-encoding nucleic acids [191,221]. However, the physicochemical interactions between CNTs and DNA have been studied under different contexts for much longer, particularly in attempts to disperse non-f -CNTs, and for the construction of sensors, nanocircuits, nanocomposites and gene delivery vectors.…”

“…An alternative functionalization reaction described by Georgakilas and coworkers produces positively charged ammonium-f -CNTs (CNT-NH 3 þ ) [146]. The interaction of CNT-NH 3 þ with plasmid DNA (pDNA) was studied in detail in our laboratories [221,233]. We have found that these f -CNTs interact with pDNA to form complexes primarily through electrostatic forces and that the pDNA was condensed around the CNT-NH 3 þ .…”

Synthesis of mesitylene-based polyamine dendrimer for functionalisation of single-walled carbon nanotubes

“…In this context, cationic and polycationic functionalised CNTs (f-CNTs) could represent a valuable alternative vector because of their capacity to easily enter into the cells to deliver plasmid DNA or short-interfering RNA. [39,40] Prato and co-workers have designed simple ammonium-f-CNTs and more sophisticated dendron-based nanotubes. [41][42] The different conjugates were used for gene-silencing experiments in vitro and in vivo demonstrating the capacity of such systems to deliver and eventually knock-down different genes efficiently.…”