## 2 Materials and Methods
### Nanomaterialssynthesis
#### 2.2.2 Lignin Extraction and LNP Synthesis  
Hazelnut tree pruning shoots (HP) and hazelnut shells (HS) were used for the synthesis of lignin nanoparticles (LNP). The lignin fractions were extracted, and LNP were synthesized from lignin-rich residues from both pruning shoots and hazelnut shells by using a solvent-antisolvent method [57; 58]. HP were collected and mechanically treated with a shredder in order to obtain a more homogeneous and defibrated matrix. Dried and cleaned HS were milled and sieved to maintain the size uniformity of powdered shells. A second physical treatment has been performed at high pressure and high temperature to make the HP and HS matrices more accessible to the following treatments: 100 g of defibrated HP and HS were firstly washed to remove the powder and environmental contaminations and put in 200 mL of distillated water and subjected to a pressure of 1.5 atm and a temperature of 121 \({}^{\circ}\)C for 30 min, by using an autoclave. The described step represents a steam explosion-like process, allowing for the opening of the biomass fibers and making them more accessible to the enzyme action. The steamed matrices (S-HP and S-HS) were dried at 60 \({}^{\circ}\)C for 24 h.  
Dried steamed matrices of HP and HS have been subjected to the enzymatic pre-treatment in order to obtain a lignin-rich biomass. Batches of 10 g of S-HP and S-HS were incubated in 100 mL of 50 mM sodium citrate buffer with a dosage of 10% and 20% _w/w_ enzyme/dried pulp, respectively. The enzymatic cocktail used was a blend of cellulases, \(\beta\)-glucosidase and hemicelluloseases (Cellic Ctec2) [59]. The enzymatic-driven depolymerization has been conducted for 96 h at 50 \({}^{\circ}\)C and 150 to 100 rpm. The action of the enzymatic cocktail is aimed to selectively depolymerize accessible reducing sugars such as cellulose and hemicellulose, and the selective release of those components allows one to obtain a residue that is rich in lignin (even though still presenting fractions of cellulose and hemicellulose). The enzymatic-treated matrices (E-HP and E-HS) were then filtered, washed from the buffer in excess and dried at 60 \({}^{\circ}\)C for 24 h. The final dried products obtained from pruning and shells residues enzymatically treated are considered as starting lignin-rich residues. The lignin fractions were extracted, and LNP were synthesized from lignin-rich residues from both pruning shoots and hazelnut shells by following the solvent-antisolvent method. The lignin-rich residues E-HP and E-HS were processed according to the protocol taken from [PERSON] et al. (2020) to obtain three different lignin fractions [60]. At first, batches of 60 g dried E-HP and E-HS were mixed with 600 mL 100% ethanol (purity \(\geq\) 99.8%, liquid, CAS No: 64-17-5) under constant stirring (300 rpm, 2 h) at room temperature. Subsequently, the insoluble and soluble fractions were separated by vacuum filtration: the insoluble residue was washed with 20 mL 100% ethanol three times, then dried, to be further processed; the soluble fraction was denoted as the L100 (dissolved in 100% ethanol) fraction, respectively, L100-HP and L100-HS. The insoluble residues were further processed by mixing with 70% ethanol, under the conditions of 300 rpm, 2 h, and room temperature. The insoluble and soluble fractions were again separated by vacuum filtration: the insoluble residue was washed with 20 mL 70% ethanol for three times, then dried, to be further processed, while the soluble fraction was denoted as the L70 (dissolved in 70% ethanol) fraction, respectively, L70-HP and L70-HS. The dried insoluble residues from the dissolution of 70% ethanol solution, denoted as the LF-HP and LF-HS fraction, were again further processed by mixing with 70% ethanol, under the conditions of 300 rpm, 2 h, and room temperature, then concentrated and directly used for the preparation of LNP, as detailed below. The ethanol used was recovered and reused for further fractionations.