Hybrid nanoparticles were prepared based on our previously reported protocol.15 (link) A mass of 50 mg of excipient-free human IgG was dissolved in 0.01 N HCl containing 20 mg of poloxamer-188 and 1 mg of enzalutamide in 1 mL with 10 mg of sodium tripoliphosphate as a carrier to make 10 mL of total solution in a 50 mL beaker. The final concentration of human IgG in each solution amounted to 5 mg/mL. This solution was then slowly titrated with 0.01 N NaOH to bring the pH of the mixture to 7, which is the isoelectric point (pI) of human IgG as determined in our laboratory using isoelectric focusing. The nanoparticles were continuously mixed on a magnetic stirrer for additional 10 min. At the pI, enzalutamide-loaded hybrid nanoparticles were spontaneously precipitated. The colloidal suspension was then centrifuged with a microcentrifuge (Eppendorf Centrifuge 5418) at 2000 rpm for 5 min. The nanoparticles were rinsed with double distilled deionized water before being redispersed in water and snapfrozen using liquid nitrogen. This was then loaded into a freezedryer (Labconco FreezeZone 4.6), and lyophilization was performed for 48 h.