Abstract
Lignin is an aromatic plant cell wall polymer that is generated in large quantities as a low-value by-product by the pulp and paper industry and by biorefineries that produce renewable fuels and chemicals from plant biomass. Lignin structure varies among plant species and as a function of the method used for its extraction from plant biomass. We first explored the impact of this variation on the physico-chemical properties of lignin nanoparticles (LNPs) produced via a solvent exchange procedure and then examined whether LNPs produced from industrial sources of lignin could be used as delivery vehicles for DNA. Spherical LNPs were formed from birch and wheat BioLignin™ and from poplar thioglycolic acid lignin after dissolving the lignin in tetrahydrofuran (THF) and dialyzing it against water. Dynamic light scattering indicated that the diameter of these LNPs was dependent on the initial concentration of the lignin, while electrophoretic light scattering indicated that the LNPs had a negative zeta potential, which became less negative as the diameter increased. The dynamics of LNP formation as a function of the initial lignin concentration varied as a function of the source of the lignin, as did the absolute value of the zeta potential. After coating the LNPs with cationic poly-l-lysine, an electrophoretic mobility shift assay indicated that DNA could adsorb to LNPs. Upon transfection of human A549 lung carcinoma basal epithelial cells with functionalized LNPs carrying plasmid DNA encoding the enhanced green fluorescent protein (eGFP), green foci were observed under the microscope, and the presence of eGFP in the transfected cells was confirmed by ELISA. The low cytotoxicity of these LNPs and the ability to tailor diameter and zeta potential make these LNPs of interest for future gene therapy applications.
Highlights
With all three lignin sources the diameter of the lignin nanoparticles (LNPs) depended on the initial concentration of lignin, whereby the diameter increased with the starting concentration (Figure 2A)
A higher initial concentration of lignin in the dialysis bag will tend to favor aggregation of lignin molecules via hydrophobic interactions during the solvent exchange, when the hydrophobic lignin is exposed to increasing concentrations of water
The range in diameter we reported (160–1194 nm) was shifted to slightly lower values than the 300–1300 nm range reported by Lievonen et al [31], which is of relevance given the greater cytotoxicity of LNPs with larger diameters
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The sinapyl three most common monolignols are p-coumaryl coniferyl plant cell and transported to the cell wall, where they can undergo oxidative polymerizaalcohol and sinapyl alcohol (Figure 1). Ten et al [29] subsequently demonstrated that these lignin nanotubes were able to deliver DNA into human (HeLa) cells without the need for auxiliary agents These LNTs were shown to be less cytotoxic compared to multi-walled carbon nanotubes, their relatively large size, with lengths in the micrometer range, may make them too large to enable the delivery of therapeutic nucleic acids to internal organs in humans or animals, due to the risk of removal by the reticuloendothelial system or via endosomal degradation [30]. This was evaluated by using LNPs as a delivery vehicle for a plasmid harboring a reporter gene to transfect A549 lung carcinoma basal epithelial cells in cell culture
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