Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment

Abstract

Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.

Introduction

Lignin is a natural aromatic biopolymer and a renewable resource obtained from lignocellulosic biomass, representing 10–15% of these materials [1]. Currently, it has been used as additive, binder, dispersant, adsorbent or surfactant [2,3]. However, the very limited solubility of the native material and the complexity of the lignin structure with very broad molecular weight distributions and a random microstructure have limited its use [4]. The functional groups, both phenolic and aliphatic hydroxyls, make it susceptible to chemical modification or polymerization, enabling the development of new materials [[5], [6], [7]]. Besides that, lignin presents some eco-friendly properties, such as biodegradability, biocompatibility and low toxicity, which make it an ideal precursor for the development of LNPs [8].

Nowadays, the development of NPs from lignin has gained interest, given its nature, for drug delivery systems [[9], [10], [11]], delivery of hydrophobic molecules [12], improvement of UV barrier [13,14], as reinforcing agent in nanocomposites [15], sorbents for heavy metal ions and other environmental pollutants, and antibacterial and antioxidant applications [[16], [17], [18], [19]]. They have also been used as an alternative to inorganic NPs due to some safety issues raised in recent years [8]. Thus, different sources of lignin have been used, being most works focused on alkali and kraft lignin [2,8,9,[20], [21], [22], [23]]. For instance, Frangville et al. [8] produced nanosized lignin particles through acid precipitation using ethylene glycol as a solvent and kraft lignin. Qian et al. [2] and Lievonen et al. [21] prepared spherical NPs from acetylated alkali lignin and kraft lignin, respectively, using THF as a solvent and the solvent displacement method. Myint et al. [24] produced LNPs using kraft lignin through compressed CO₂ antisolvent method. LPs based on organosolv lignin were produced by Richter et al. [25] and Liu et al. [26], using acetone and tetrahydrofuran as a solvent, respectively, through the solvent displacement method. Most of these published production methods have some drawbacks, including extensive use of organic solvents that poses a potential hazard to the environment, irregular shapes and difficulty to control the particle size and size distribution.

In the current work, lignin particles were produced from organosolv lignin, using ethanol as solvent through the solvent displacement method by dripping the lignin solution in an antisolvent (water). Matsakas et al. [27,28] also reported the production of LNPs from organosolv lignin solubilized in ethanol by the solvent displacement method through the solvent evaporation or by adding an antisolvent via dialysis or dilution. This approach is interesting since it avoid some of the drawbacks described above, such as toxic and expensive solvents and particles with irregular shapes. Ethanol is considered a green, safer and cost-effective solvent and is completely miscible with water.

In 2011, the EC adopted a new definition for nanomaterial [29], referred in this work as ‘EC NM definition’, which refers ‘Nanomaterial’ as a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where 50% or more particles (number size distribution) have one or more external dimensions in the size range of 1 nm–100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%. By other side, the FDA mentions that in some cases material with sizes up to 1000 nm should also be considered nanomaterial, justified by the fact “at the present time, available scientific information does not establish a uniform upper boundary above 100 nm where novel properties and phenomena similar to those seen in materials with dimensions in the nanoscale range cease for all potential materials or end products” [30]. Based on this, materials with sizes higher than 100 nm should be evaluated in terms of their properties and safety, such as micro and nanostructures based on biomacromolecules, that due to their complexity should be evaluated case-by-case. In the case of lignin, this has never been addressed and the advantages and challenges of using lignin as a nanomaterial when compared with micro size are not fully understood.

Thus, this study reports the production of lignin particles (LPs) with different size scales (nano and micro), according to the EC NM definition, using a green synthesis process and correlates their main properties, including the possible cytotoxicity. LPs were characterized by dynamic light scattering and transmission electron microscopy. Their chemical structure and thermal stability were evaluated by FTIR, TGA and DSC. Moreover, bioactive properties of LPs were evaluated through antioxidant and antimicrobial tests. Finally, the in vitro cytotoxicity and the cellular antioxidant activity of LPs were assessed using a Caco-2 cell line.