**4.1 Techniques used for the synthesis of LNP**

Kumar et al. [58] present an interesting resumé of the several strategies used to produce lignin micro and nanoparticles. Diverse techniques can be used and include: shifting methods (by pH or solvents) [97], a template-based synthesis technique (or polymerization), aerosol process, electrospinning, supercritical fluid processes, solvent antisolvent precipitation [98], or acoustic cavitation. A resumé of techniques to attain LNP is presented below.

**Self-assembly**—is the most common method to produce LNP; it starts by dissolving the lignin in an organic solvent and by adding the antisolvent (usually water) or, during dialysis, the particles start to form in a self-assembling process.

**Antisolvent precipitation**—first the lignin is dissolved in a solvent, such as: acetone solution (9:1, v/v) [99]; tetrahydrofuran (THF) [62]; dimethylsulfoxide (DMSO) [92]; dimethylformamide (DMF) [100]; and aqueous sodium *p*-toluenesulfonate (APS) [101]; then this solution is added slowly to an antisolvent (usually water, where the lignin is insoluble) to form the LNP. It is important to choose properly the solvents and antisolvents for the formation of LNP; good miscibility between the lignin and the solvent is the best choice. Solvents with good miscibility and a low boiling point lead to the formation of smaller size LNP and with a spherical shape, but if the solvent has poor miscibility and the antisolvent has a higher boiling point, the resulting LNPs do not have a uniform shape and are larger [58]. But other parameters such as the lignin concentration in the solvent, antisolvent temperature, and stirring rate are also important [58].

**pH shifting**—this technique has similarities with the posterior technique; but here the pH is changed from acid to base or vice versa. Lignin solubility changes with the pH of the solution, and precipitates. As examples of these methods: (i) the lignin is dissolved in NaOH at pH 12 and by HNO3 the LNPs precipitate [102]; (ii) the lignin is dissolved in ethylene glycol and the LNPs are precipitated by adding hydrochloric acid [95]. The use of an acid to precipitate the LNP makes the process more expensive and dangerous, but the acid can be recovered, and the method has the advantage of attaining well-defined LNP with a good shape and stability [58].

**Solvent exchange or solvent shifting**—this technique involves a dual system of solvents, an organic solvent (where the lignin is dissolved), and water (in excess allowing the formation of LNP due to the decrease of lignin solubility in water at pH neutral). The LNP size ranges from nanometer to hundreds of micrometers with spherical-shaped particles [58]. Suitable solvents are: THF, acetone, acetone/water, and DMSO [58]. This is similar to the antisolvent precipitation methods, but it is the excess of water that makes the LNP; in the other method, it was the pH shifting. For the solvent exchange method, the dissolved lignin is put in dialysis bags immersed in water and the LNPs are formed during the dialysis process. LNPs were produced by dialysis of a mixture of lignin solution and oleic acid coated with Fe3O4 [62]. The need for dialysis bags increases the costs of this method (**Table 8**).

**Supercritical fluid** (SCF)—this is a technique widely used in the pharmaceutical industry. Generally, the supercritical fluid used is CO2 due to its diverse advantages (abundance, nontoxicity, low cost, and not flammable). This technique has different names depending on the conditions and the fluids used: supercritical antisolvent (SAS), supercritical gas antisolvent (GAS) are just two examples; LNPs were prepared by SAS, using acetone (solvent) and CO2 (antisolvent) from poplar organosolv lignin; because the solubility was enhanced, the LNP formed (0.144 μm) presented higher antioxidant capacities compared with the starting lignin [103].


#### **Table 8.**

*Some advantages and disadvantages of the methods used for the synthesis of LNP. Adapted from Schneider et al. [60] and Kumar et al. [58].*

**Aerosol process**—this is a single-step method for the production of nanoparticles applied in biomedical applications and was first presented by Eerikäinen et al. [104]; it consists of an atomizer that works continuously to generate an aerosol. This aerosol passes through a heated tube connected to a low-pressure impactor where the particles formed are collected. Researchers were successfully able to produce LNPs with sizes ranging from 30 to 2000 nm [105].

**Ultrasonication or acoustic cavitation**—the lignin is an aqueous solution and this suspension is sonicated by changing the intensity, time, and temperature; then the sample is dried under mild conditions [106]. These authors used two commercial alkali lignins suspensions to produce efficiently nanosized particles (spherical shapes

and homogeneous). If this technique is used alone, the LNPs formed are not uniform in terms of size or the particle size distribution, which are dependent on the ultrasound conditions. Some researchers have overcome this by producing the LNP by solvent exchange combined with ultrasound. This combination is advantageous because the LNPs are rapidly formed and easily detached by centrifugation.

**Electrospinning**—this is a popular technique for the production of nanomaterials; the lignin is dissolved in a suitable solvent and the mixture is put in a syringe, pumped through a nozzle with reduced size (diameter of around 100 μm) that jets the liquid into a collector plate, creating a nanofiber matrix; an electric field is used with voltage of 100–500 kV/m to create the nanofibers (10–25 cm). Technical lignins (softwood Kraft, hardwood Kraft, and sulfonated Kraft lignins) were mixed with PEO (polyethylene oxide) at different percentages (1–5 wt%) and obtained fibers by electrospinning [107]; the incorporation of PEO improved the fibers produced (more uniformity) at lower viscosities. However, this technique has limitations associated with the lignin properties and it was suggested that electrospraying would be more favorable for nanoparticles production [58].

**Interfacial crosslinking**—this is not a method for LNP, but is better for their stabilization, generating particles and capsules. The process involves lignin emulsification, usually in an oil-water phase, where the addition of a cross-linking agent improves the lignin cross-link. First, the lignin is dissolved in an alkaline solution that will activate the OH chains and generate a dispersed phase; or it will be dissolved in a solvent with a surfactant such as Span80®, 1-pentanol, or Tween 80®. An oil/water microemulsion is prepared and emulsified to the solubilized lignin, creating the lignin cross-linkages with the water/oil interface. The next step is to add the cross-linking agent, which can be toluene diisocyanate, thiol, or epichlorohydrin [60].

**Polymerization**—this is similar to the cross-linking method. The lignin has to be modified or some groups activated so that they can be grafted onto a surface that has an affinity to the lignin. As examples of the application of this technique: lignin nanotubes were produced in an alumina membrane that was dissolved in phosphoric acid [108]; lignin nanoparticles were produced by lignin being grafted onto PDMAEMA (2-(dimethylamino)ethyl-methacrylate) and PDEAEMA (2-(diethylamino)ethyl methacrylate) [109, 110].

**Biological pathway**—this is not a common technique for LNP synthesis, so far. LNPs are produced by microorganisms or by enzymes (formed by either bacteria or fungi). Examples: (i) enzymes were used to break the linkages between lignin and cellulose in *Luffa* fibers, producing lignin-derived particles with cuboidal shape and size around 20–100 nm [111]; (ii) lignin was extracted by coconut fibers (soda pulping), the lignin obtained was hydrolyzed using *Aspergillus* sp. Nanolignin was synthetized by different techniques at distinct yields: homogenization (81%), ultrasonication (64.3%), and microbial (58.4%) [76]; (iii) the colloidal stability of LNP was improved using fungi species [87].

**Table 8** presents some of the methods mentioned and their strengths, as well as some of their weaknesses. The comparison was made regarding the type of solvents used (if environmentally-safe, cost), but also regarding the process time, LNP morphology, and size. Generally, solvents such as acetone, dioxane, THF, and DMSO are used in medium volume, purchased, and are relatively affordable. The use of hazardous reagents is a negative point. Some studies include green solvents such as levulinic acid that had high efficiency in dissolving Kraft lignin (up to 40%) compared with other carboxylic acids, and the lignin dissolved can be regenerated by adding an





**Table 9.**

*Compilation of some potential uses and methods for lignin nanoparticles production.*

excess of water, maintaining the morphological characteristics and the thermal stability of the lignin [112].

Researchers are able to produce LNP with a range of shapes, such as nanoparticles [62–64], nanotubes [108], nanospheres [113], nanofibers [114], nanocapsules [115], nanomicelles [85], nanofilms [116]; and sizes (micrometers and nanometers). This high diversity of shapes and sizes contrasts with a high demand for uniformity for

specific applications, which requires reproducibility at industrial-scale production [58]. According to Zhang et al. [43], within a certain lignin concentration range, the particle size increased with increasing lignin concentration. A higher concentration of lignin in the system means more lignin available for the growth of the nanospheres (LNS); a high stirring rate of aqueous phase and ionic liquids produces smaller particles. Also, the dropping speed of water affects the diameters of the LNS, where the increase of the water dropping speed from 2 to 6 mL/s decreased the LNS diameter [117]. Besides these aspects, the feasibility of LNP production depends on the scale-up, manufacturing costs, and applications. Having this in mind, Assis and coworkers [118] carried out a techno-economic study to evaluate the costs of lignin production, the costs of LNP synthesis, and their applications. **Table 9** presents a list of LNPs produced by different methods and compiled by their possible applications.
