Preparation of Silk Fibroin Nanoparticles

The preparation of SFNs was based on the method described previously by Lozano-Pérez et al. [66 (link)], with modifications. Briefly, an SF-ionic liquid (SIL) solution (10 wt %) was prepared by adding 0.5 g of SF to 4.5 g of [emim⁺][CH₃COO⁻]. The mixture was treated with a 3/8″ tapered horn of a Sonifier Branson 450D (Emmerson Ultrasonic Corporation, Danbury, CT, USA), with pulsating ultrasonication steps at 30% amplitude at a controlled temperature below 90 °C until complete dissolution. To this solution freshly prepared, 3 mL of ultrapure water was slowly added to reduce viscosity. The final concentration of the SIL solution after diluting with 3 mL of ultrapure water was 6.66 wt %. After heating to 60 °C, the SIL solution was propelled using a peristaltic pump and then sprayed onto 100 mL of gently stirred methanol at −20 °C by a thermostatically controlled 0.7 mm two-fluid nozzle (from a Mini Spray Dryer B-290, BÜCHI Labortechnik, Flawil, Switzerland, Part No. 044698) which uses compressed N₂ to disperse the solution into fine droplets. A milky white suspension appeared and the suspension was allowed to reach room temperature while stirring for 2 h. Then, the nanoparticle suspension was transferred to centrifuge vials and centrifuged at 13,400 rpm for 15 min, at 4 °C (Sigma 3-18K Centrifuge with a 19,776 H angle rotor, Osterode, Germany). The supernatant, which is free of nanoparticles, was removed and reserved for subsequent recycling of the ionic liquid. An equal volume of fresh methanol was added to the vial, and the white precipitate was suspended by vigorous stirring in a vortex mixer for 2 min and 5 min of ultrasonication with a Branson 450D sonicator (Emmerson Ultrasonic Corporation, Danbury, CT, USA). The centrifugation step was repeated under the same conditions. The white precipitate was subjected to successive rinses with ultrapure water to remove the remaining methanol and ionic liquid. The particles were lyophilized in an Edwards Modulyo 4K Freeze Dryer (Thermo Scientific, Waltham, MA, USA) for 72 h, at −55 °C and 0.5 mbar to obtain dry particles. The methanolic fractions were mixed before recovery of the ionic liquid by removing of the methanol/water on a BÜCHI RE-111 rotary evaporator (Flawil, Switzerland) at 80 °C and 80 mbar. The ionic liquids were kept in a desiccator until reuse.