Novel trimethyl lock based enzyme switch for the self-assembly and disassembly of gold nanoparticlesw

The programmed self-assembly of gold nanoparticles (AuNPs) with controlled surface chemistry has attracted considerable attention for their potential applications in drug delivery and medical diagnosis. A number of specific biomolecular recognition motifs such as complementary oligonucleotide hybridization, biotin-avidin binding and enzymatic catalytic reactions have been extensively exploited for the control of AuNPs assembly, which provide a simple and specific sensing platform for the systematic identification of a variety of molecular analytes including DNAs, bacterial toxins, proteins and enzymes by performing colorimetric or surface enhanced Raman scattering (SERS) measurements. However, most of the molecular recognitions were mainly based on one-step AuNPs aggregation or dispersion and these assembly or disassembly processes were usually achieved in a separate population of gold nanoparticles. A few recognition processes with various degrees of continuous two-step self-assembly of AuNPs have been reported, in which the triggered changes in their assembled states were driven by a physical or chemical perturbation such as pH, temperature, light, concentration of inorganic/organic molecules, or fuelling oligonucleotides. However, the development of AuNPs network where the self-assembly and disassembly are associated with multiple enzyme stimuli within one population of nanoparticles has not been well-exploited yet. In this study, we present a novel ‘‘trimethyl lock’’ based dual enzyme-responsive AuNPs conjugate which can be utilized to control the self-assembly and disassembly of AuNPs in the same population of nanoparticles (Scheme 1). The enzymeresponsive AuNPs conjugate in our design consists of two sections: (1). A unique ‘‘trimethyl lock’’ lactonization section to release the peptide linker upon the esterase treatment, leading to the self-assembly of AuNPs; (2). A protease active section to cleave the peptide and induce further AuNPs disassembly (Scheme 2). Both esterase and protease are known to be abundant in nature and are essential for many important biological processes, and they are also involved in various disease states such as HIV, cancer, Alzheimer’s and heart diseases. Based on the specific enzymatic hydrolysis, both the self-assembly and disassembly of AuNPs can be achieved in the same nanoparticles system and this sequential two-step process will be easily monitored by naked eye, simple spectrophotometer and surface enhanced Raman scattering (SERS) measurements. To demonstrate the proof of concept, we designed the selfassembled functionalized AuNPs network based on dual enzyme reactions. As shown in Scheme 2, a flexible 4-mercaptophenylacetic acid modified thermolysin cleavable peptide (SH–Ph–CH2–Gly–Gly–GlykPhe–Gly–Gly–Lys(NH2)–CO– NH2) was connected to the carboxyl on the acetylated o-hydroxyphenolpropionic acids derivatives (See the ESIw), which are well known as ‘‘trimethyl lock’’ commonly used in ‘‘prodrug’’ development and fluorescent imaging, to afford the thiol ester and amide bond at each end of the peptide sequence. The advantage of introducing ‘‘trimethyl lock’’ in this investigation is to achieve the effective esterase hydrolysis for the further release of the peptide linker based on lactonization reaction, and also to significantly minimize the selfaggregation of AuNPs caused by enzyme substrate itself as reported in previous study. Typically, the ‘‘trimethyl lock’’ peptide substrate (8.5 mM) was firstly incubated with esterase (1.25 mg mL ) in monodispersed bis (p-sulfonatophenyl) phenylphosphane stabilized AuNPs suspension (pH 7.4) for 2 h. The enzyme hydrolysis catalyzed by esterase resulted in the cleavage of the thiolester bond and removal of acetyl group from the ‘‘trimethyl lock’’ peptide substrate. Unmasking of phenolic oxygen would facilitate the lactone formation