Controlled Release of Guest Molecules from Spherical Assembly of Trigonal Gultathione by Disulfide Recombination Kazunori Matsuura,* 1,2 Keigo Tochio, 1 Kenta Watanabe, 1 and Nobuo Kimizuka 1,2 1 Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395 2 International Research Center for Molecular Systems (IRCMS), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395 (Received April 12, 2011; CL-110304; E-mail: ma2ra-k@mail.cstm.kyushu-u.ac.jp) Nanospheres formed by self-assemblyof trigonal gluta- thione (TG) underwent disulfide recombination upon addition of dithiothreitol. Guest molecules encapsulated in the nanospheres were gradually released during the recombination process. In biological systems, many redox reactions involving disulfide bonds play pivotal roles in protein folding and consequently in the control of protein functions. 1 Since cytoplasm is generally a reductive environment, many disulfide bonds exist as the reduced form (thiol group) in cells. In the past decade, many methodologies for controlled release of drugs based on the cleavage of disulfide bonds have been devel- oped. 24 For example, release of nucleic acids 2 and drugs 3 from polymer micelles has been regulated by the reduction of disulfide bonds. Controlled gating systems to release drugs from mesoporous silica using the redox reaction of disulfide bonds have been also reported. 4 It has become feasible to reversibly control the structures of polymers or nanoassemblies by applying dynamic covalent bonds such as disulfide and Schiff base. 59 Recently, Kataoka and co-workers reported a structural transition from polyion complex micelles bearing poly(ethylene glycol) into polyion complex polymersomes (PICsome) by reduction of disulfides. 8 Otsuka and co-workers reported chain recombination of disul- fide-containing polyesters by photoirradiation. 9 Whereas the controlled release of guest molecules based on the reductive cleavage of disulfide bonds has been widely studied as mentioned above, structural transition of nanoassemblies by recombination of disulfides and their application to controlled molecular release are much less common. We have developed C 3 -symmetric peptide conjugates mimicking spherical viruses and clathrin and reported their self-assembling behavior in water. 1012 It has been reported that a C 3 -symmetric conjugate bearing glutathione (trigonal gluta- thione, TG), which is an ubiquitous natural tripeptide, sponta- neously self-assembled into 100250 nm nanospheres in water and that uranine was encapsulated into the nanosphere. 11a In this paper, we report controlled release of guest molecules from the TG assemblies by disulfide recombination (Figure 1). TG was synthesized from trimesoyl chloride according to our previous report, and its chemical structure was confirmed by MALDI-TOF-MS and 1 H NMR. 11a TG was dissolved in 10 mM Tris-HCl buffer (pH 7) without heating and sonication. After the TG solution was equilibrated at 25 °C for at least 30 min, the self-assembling behavior in the buffer was investigated. The dynamic light scattering (DLS) of 0.33 mM TG in the buffer showed formation of monodispersed assemblies with the size of 68 « 13 nm (Figure 2A). 13 The SEM also showed the existence of 50100 nm spherical assemblies (Figure 2B). The TG assemblies in 10mM Tris-HCl buffer (pH 7) were smaller than those in pure water (173 « 32 nm) at the same concentration (Figure S1 16 ). The observed shrinkage of TG assemblies is likely to be caused by the electrostatic interaction between Tris- HCl cations and anionic TG.Figures 2C and 2D, respectively, show size distribution obtained from DLS and SEM images of the TG assemblies two days after addition of 1.0 mM dithio- threitol (DTT, 1 equivalent to disulfide bonds of TG). Nano- particles with average diameter of 72 « 15 nm were observed, with a small fraction of these with increased diameter of 234 « 73 nm. The large particles might be caused by the fusion among TG assemblies through the formation ofinterparticle disulfide bonds. Figure 3 shows the time course of reversed phase HPLC observed for the reaction of TG and DTT in 10mM Tris-HCl Figure 1. Schematic illustration of disulfide recombination in spherical assemblyof trigonal glutathione (TG). Published on the web June 4, 2011 711 doi:10.1246/cl.2011.711 © 2011 The Chemical Society of Japan Chem. Lett. 2011, 40, 711713 www.csj.jp/journals/chem-lett/