Nano-encapsulation of scFv Protein

scFv at 1% w/w was encapsulated into 10:90 CPTEG:SA NPs by an anti-solvent nano-encapsulation method at room temperature, as described previously [41 (link)]. Briefly, scFv at 20 mg/mL in 50 µL PBS was added to non-functionalized polymer dissolved in methylene chloride at 20 mg/mL while sonicating at 30% for 30s and immediately precipitated into pentane (Thermo Fisher Scientific) at room temperature at a 1:250 MeCl₂: pentane ratio. NP morphology and size were determined using field-emission scanning electron microscopy (Quanta 250 FEG, Hillsboro, OR, USA). ImageJ 1.43u software (National Institutes of Health, Bethesda, MD, USA) was utilized to quantify NP size and size distribution. NP release kinetics and encapsulation efficiency were performed in vitro as described previously [43 (link)]. Briefly, scFv total protein was determined using a Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), and the absorbance was recorded using a spectrophotometer at OD570. A standard curve of scFv from 0.5–125 µg/mL was used for quantification.