Lignin Nanotubes As Vehicles for Gene Delivery into Human Cells

Lignin
nanotubes (LNTs) synthesized from the aromatic plant cell
wall polymer lignin in a sacrificial alumina membrane template have
as useful features their flexibility, ease of functionalization due
to the availability of many functional groups, label-free detection
by autofluorescence, and customizable optical properties. In this
report we show that the physicochemical properties of LNTs can be
varied over a wide range to match requirements for specific applications
by using lignin with different subunit composition, a function of
plant species and genotype, and by choosing the lignin isolation method
(thioglycolic acid, phosphoric acid, sulfuric acid (Klason), sodium
hydroxide lignin), which influences the size and reactivity of the
lignin fragments. Cytotoxicity studies with human HeLa cells showed
that concentrations of up to 90 mg/mL are tolerated, which is a 10-fold
higher concentration than observed for single- or multiwalled carbon
nanotubes (CNTs). Confocal microscopy imaging revealed that all LNT
formulations enter HeLa cells without auxiliary agents and that LNTs
made from NaOH-lignin penetrate the cell nucleus. We further show
that DNA can adsorb to LNTs. Consequently, exposure of HeLa cells
to LNTs coated with DNA encoding the green fluorescent protein (GFP)
leads to transfection and expression of GFP. The highest transfection
efficiency was obtained with LNTs made from NaOH-lignin due to a combination
of high DNA binding capacity and DNA delivery directly into the nucleus.
These combined features of LNTs make LNTs attractive as smart delivery
vehicles of DNA without the cytotoxicity associated with CNTs or the
immunogenicity of viral vectors.