Silk fibroin (SF) is a natural biopolymer that has been extensively studied in various applications due to its impressive mechanical properties and biocompatibility. Recently, SF-based particles have been proposed as controlled drug delivery systems. A new and efficient method to prepare SF microemulsions (SF-MEs) was developed by oil-in-water emulsions using high-pressure homogenization to promote emulsification. During SF-ME production, the secondary structure of SF changed to a more stable conformation (from random coil to -sheets), thus allowing the formation of small and stable (140.7 ± 1.9 nm; polydispersity index, 0.25) SF microparticles (SF-MPs). The efficiency of SF-MP formation was 60%. Orange IV was used as a model compound for incorporation and release studies, although its incorporation into the SF-MEs significantly improved particle size and size distribution over at least 4 wk compared to traditional stabilizers (e.g., poloxamer 407, transcutol, Tween 80, and SDS). This should be a call of attention when using dyes as model compounds since they can influence particle properties and lead to misinterpretation of the results. Orange IV showed an incorporation efficiency of 91% and a controlled release over time. Stable SF-MP formulations, further enhanced by orange IV incorporation, provide an innovative method with potential application in pharmaceutical development due to its associated high biocompatibility and release profile.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the project "BioHealth - Biotechnology and Bioengineering Approaches to Improve Health Quality," Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. We would also like to acknowledge CRA - Unita di Ricerca di Apicoltura and Bachicoltura Padova (Italy) for the supply of B. mori silkworm cocoons, and Andreia Vasconcelos for technical support
