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Protein-loaded dextran nanoparticles were prepared by the method of freezing-induced phase separation. The average diameter of protein-loaded dextran nanoparticles prepared using this method was between 100 and 300 nm. The protein-loaded dextran nanoparticles were then microencapsulated into polylactide-co-glycolide acid (PLGA) microspheres by the solid-in-oil-in-water (S O W) multiple emulsion technique. The release behaviour of the microspheres showed that the method might improve the release profile and bioactivity in vitro, and also reduce initial burst and aggregation. The method will be potentially developed to solve protein-controlled release problems.