DOI: 10.1002/chem.200902098 Microspheres of Mixed Proteins Ulyana Angel (Shimanovich),* [a, b] Devorah Matas, [b] Shulamit Michaeli, [b] Artur Cavaco-Paulo, [c] and Aharon Gedanken* [a] Introduction Ultrasonic emulsification is a well-known process and occurs in biphasic systems. Emulsification is necessary for microcapsule formation. Protein microspheres have a wide range of applications, including drug and oxygen delivery systems, [2–3, 12] contrast agents for sonography, [4] and MRI. [5–7] Micrometer-sized air-filled or liquid-filled proteinaceous microspheres (PMs) were synthesized from various kinds of proteins, such as bovine serum albumin (BSA), [8–10] human serum albumin (HAS), [11] and hemoglobin (Hb) [12] by the so- nochemical method developed by Suslick and co-workers. In the late 1960s, a modified polymerization method for the preparation of proteinaceous microspheres was developed by Rhodes, Scheffel, Wagner, and Zolle et al. [13–15] The mi- crosphere formation was accomplished by either heat dena- turation at various temperatures or by cross-linking with car- bonyl compounds in the ether phase. Other cross-linking agents, such as glutaraldehyde, were also used. Furthermore, air-filled human serum albumin microspheres were made by Dick and Feinstein [16, 17] as contrast agents in echosonogra- phy. However, these methods yielded microspheres with a short storage life, low microbubble stability, or high toxicity. The first liquid-filled proteinaceous microspheres were pre- pared by Suslick. They were made of BSA and were filled with n-dodecane, n-decane, n-hexane, cyclohexane, or tolu- ene. The synthesis was conducted under a high-intensity ul- trasonic probe, and 1.5  10 9 microcapsules per mL were ob- tained upon sonicating the precursor solution under air or O 2 . The average diameter of the PM was 2.5 mm with a narrow size distribution (Gaussian distribution =  l.0 mm). The mechanism of the sonochemical formation of PM has been discussed previously. [18] According to this mechanism, the microspheres are formed by chemically cross-linking cys- teine residues of the protein with HO 2 radicals formed around a micron-sized gas bubble or a nonaqueous droplet. The chemical cross-linking is responsible for the formation of the microspheres, and is a direct result of the chemical ef- fects of ultrasound radiation on an aqueous medium. In the current fundamental studies, we have extended Suslicks method and applied it to more than one protein, Abstract: This paper describes the syn- thesis of mixed proteinaceous micro- spheres (MPMs) by the sonochemical method. The current fundamental re- search follows the research of Suslick and co-workers who have developed a method by which high-intensity ultra- sound is used to make aqueous suspen- sions of proteinaceous microcapsules filled with water-insoluble liquids. [1] By using high-intensity ultrasound, we have synthesized microspheres made of a few different proteins. The three pro- teins used in the current experiments are bovine serum albumin (BSA), green fluorescent protein (GFP), and cyan fluorescent protein–glucose bind- ing protein–yellow fluorescent fused protein (CFP-GBP-YFP). The two syn- thesized microspheres made of mixed proteins are BSA-GFP and BSA-(CFP- GBP-YFP). This paper presents the characterization of the sonochemically produced microspheres of mixed pro- teins. It also provides an estimate of the efficiency of the sonochemical pro- cess in converting the native proteins to microspheres. Keywords: core–shell structures · micelles · proteins · sonochemistry · surface modification [a] U. Angel (Shimanovich), Prof. A. Gedanken Department of Chemistry and Kanbar Laboratory for Nanomaterials, Bar-Ilan University Center for Advanced Materials and Nanotechnology Bar-Ilan University, Ramat-Gan 52900 (Israel) Fax: (+ 972) 3-7384053 E-mail : gedanken@mail.biu.ac.il [b] U. Angel(Shimanovich), Dr. D. Matas, Prof. S. Michaeli Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan, 52900 (Israel) Fax: (+ 972)3-7384053 [c] Prof. A. Cavaco-Paulo Textile Engineering Department University of Minho, 4800058 Guimaraes (Portugal) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200902098.  2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Chem. Eur. J. 2010, 16, 2108 – 2114 2108