Formation of Catalytic Hotspots in ATP-Templated Assemblies.

The self-assembly of surfactant-based structures that rely for their formation on the combination of a thermodynamically controlled and a dissipative pathway is described. Adenosine triphosphate (ATP) acts as a high-affinity template and triggers assembly formation at low surfactant concentrations. The presence of these assemblies creates the conditions for the activation of a dissipative self-assembly process by a weak-affinity substrate. The substrate-induced recruitment of additional surfactants leads to the spontaneous formation of catalytic hotspots in the ATP-stabilized assemblies that cleave the substrate. As a result of the two self-assembly processes, catalysis can be observed at a surfactant concentration at which low catalytic activity is observed in the absence of ATP.

Exploiting multivalency and cooperativity of gold nanoparticles for binding phosphatidylinositol (3,4,5)-trisphosphate at sub-nanomolar concentrations.

Cationic, monolayer-protected gold nanoparticles provide a multivalent charged surface and a hydrophobic monolayer that synergistically contribute to the binding of phosphatidylinositol (3,4,5)-trisphosphate, a relevant biomarker. The observed dissociation constant is in the picomolar region, providing the possibility of using these gold nanoparticles for the selective extraction of this molecule from biological fluids.

The Mechanism of Cleavage of RNA Phosphodiesters by a Gold Nanoparticle Nanozyme.

The cleavage of uridine 3'-phosphodiesters bearing alcohols with pKa ranging from 7.14 to 14.5 catalyzed by AuNPs functionalized with 1,4,7-triazacyclononane-Zn(II) complexes has been studied to unravel the source of catalysis by these nanosystems (nanozymes). The results have been compared with those obtained with two Zn(II) dinuclear catalysts for which the mechanism is fairly understood. Binding to the Zn(II) ions by the substrate and the uracil of uridine was observed. The latter leads to inhibition of the process and formation of less productive binding complexes than in the absence of the nucleobase. The nanozyme operates with these substrates mostly via a nucleophilic mechanism with little stabilization of the pentacoordinated phosphorane and moderate assistance in leaving group departure. This is attributed to a decrease of binding strength of the substrate to the catalytic site in reaching the transition state due to an unfavorable binding mode with the uracil. The nanozyme favors substrates with better leaving groups than the less acidic ones.

A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex.

Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface-passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids-processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pKa of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis-p-nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

Host-Guest Allosteric Control of an Artificial Phosphatase.

The activity of many enzymes is regulated by associative processes. To model this mechanism, we report here that the conformation of an unstructured bimetallic Zn(II) complex can be controlled by its inclusion in the cavity of a γ-cyclodextrin. This results in the formation of a catalytic bimetallic site for the hydrolytic cleavage of the RNA model substrate HPNP, whose reactivity is 30-fold larger with respect to the unstructured complex. Competitive inhibition with 1-adamantanecarboxylate displaces the metal complex from the cyclodextrin decreasing the reactivity.

Oligopeptide Helical Conformations Control Gold Nanoparticle Cross-Linking.

Peptide sequences functionalized with primary amines at the N- and C-terminus are able to induce the aggregation of gold nanoparticles in ethanol as a consequence of their folding into a helical conformation. Random coil peptides are unable to induce such an aggregation process. Aggregation can be monitored spectrophotometrically by following the shift of the surface plasmon resonance (SPR) band of the nanoparticles and is confirmed by transmission electron microscopy and dynamic light scattering analyses. Partial denaturation of the peptides results in diminished cross-linking ability. The helicity parameter θ222 /θ208 correlates fairly well with the shift of the SPR band to longer wavelengths, supporting the relationship between the amount of helical content of a peptide sequence and its ability to induce aggregation.

Factors Influencing the Activity of Nanozymes in the Cleavage of an RNA Model Substrate.

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis-Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium.

Gold nanoparticles crosslinking by peptides and amino acids: A tool for the colorimetric identification of amino acids.

Gold nanoparticles are known to aggregate in the presence of proper multifunctional compounds. For those larger than 3 nm, the process can be followed with the naked eye because the surface plasmon absorption band of the particles shifts to longer wavelengths and the solution color changes from red to blue. To exploit this property, we have used amino acids and peptides as crosslinking agents in ethanol at low µM concentrations. In the case of amino acids, we report a straightforward protocol for their colorimetric identification. We also report that the presence of precise secondary structure and conformational constraints in oligopeptides, directly influence their crosslinking ability. A discussion on the nature of the interactions that induce these phenomena is also reported.

Fuel-Selective Transient Activation of Nanosystems for Signal Generation.

The transient activation of function using chemical fuels is common in nature, but much less in synthetic systems. Progress towards the development of systems with a complexity similar to that of natural ones requires chemical fuel selectivity. Here, we show that a self-assembled nanosystem, composed of monolayer-protected gold nanoparticles and a fluorogenic peptide, is activated for transient signal generation only in case the chemical fuel matches the recognition site present at the nanoparticle surface. A modification of the recognition site in the nanosystem completely changes the chemical fuel selectivity. When two nanosystems are simultaneously present, the selectivity expressed by the system depends on the concentration of nucleotide added.

Binding and Uptake into Human Hepatocellular Carcinoma Cells of Peptide-Functionalized Gold Nanoparticles.

One of the most daunting challenges of nanomedicine is the finding of appropriate targeting agents to deliver suitable payloads precisely to cells affected by malignancies. Even more complex is the ability to ensure that the nanosystems enter those cells. Here, we use 2 nm (metal core) gold nanoparticles to target human hepatocellular carcinoma (HepG2) cells stably transfected with the SERPINB3 (SB3) protein. The nanoparticles were coated with a 85:15 mixture of thiols featuring, respectively, a phosphoryl choline (to ensure water solubility and biocompatibility) and a 28-mer peptide corresponding to the amino acid sequence 21-47 of the hepatitis B virus-PreS1 protein (PreS1(21-47)). Conjugation of the peptide was performed via the maleimide-thiol reaction in methanol, allowing the use of a limited amount of the targeting molecule. This is an efficient procedure also in the perspective of selecting libraries of new targeting agents. The rationale behind the selection of the peptide is that SB3, which is undetectable in normal hepatocytes, is overexpressed in hepatocellular carcinoma and in hepatoblastoma and has been proposed as a target of the hepatitis B virus (HBV). For the latter, the key recognition element is the PreS1(21-47) peptide, which is a fragment of one of the proteins composing the viral envelope. The ability of the conjugated nanoparticles to bind the target protein SB3, expressed in liver cancer cells, was investigated by surface plasmon resonance analysis and in vitro via cellular uptake analysis followed by atomic absorption analysis of digested samples. The results showed that the PreS1(21-47) peptide is a suitable targeting agent for cells overexpressing the SB3 protein. Even more important is the evidence that the gold nanoparticles are internalized by the cells. The comparison between the surface plasmon resonance analysis and the cellular uptake studies suggests that the presentation of the protein on the cell surface is critical for efficient recognition.

Chiral Nanozymes-Gold Nanoparticle-Based Transphosphorylation Catalysts Capable of Enantiomeric Discrimination.

Enantioselectivity in RNA cleavage by a synthetic metalloenzyme has been demonstrated for the first time. Thiols containing chiral Zn(II) -binding head groups have been self-assembled on the surface of gold nanoparticles. This results in the spontaneous formation of chiral bimetallic catalytic sites that display different activities (kcat ) towards the enantiomers of an RNA model substrate. Substrate selectivity is observed when the nanozyme is applied to the cleavage of the dinucleotides UpU, GpG, ApA, and CpC, and remarkable differences in reactivity are observed for the cleavage of the enantiomerically pure dinucleotide UpU.

Hydrolytic Metallo-Nanozymes: From Micelles and Vesicles to Gold Nanoparticles.

Although the term nanozymes was coined by us in 2004 to highlight the enzyme-like properties of gold nanoparticles passivated with a monolayer of Zn(II)-complexes in the cleavage of phosphate diesters, systems resembling those metallo-nanoparticles, like micelles and vesicles, have been the subject of investigation since the mid-eighties of the last century. This paper reviews what has been done in the field and compares the different nanosystems highlighting the source of catalysis and frequent misconceptions found in the literature.

Helical peptide-polyamine and -polyether conjugates as synthetic ionophores.

Two new synthetic ionophores in which the hydrophobic portion is represented by a short helical Aib-peptide (Aib=α-amino-isobutyric acid) and the hydrophilic one is a poly-amino (1a) or a polyether (1b) chain have been prepared. The two conjugates show a high ionophoric activity in phospholipid membranes being able to efficiently dissipate a pH gradient and, in the case of 1b, to transport Na(+) across the membrane. Bioactivity evaluation of the two conjugates shows that 1a has a moderate antimicrobial activity against a broad spectrum of microorganisms and it is able to permeabilize the inner and the outer membrane of Escherichia coli cells.

Efficient phosphodiester cleaving nanozymes resulting from multivalency and local medium polarity control.

The self-organization of Zn(II) complexes on the surface of 1.6-nm diameter gold nanoparticles (nanozymes) allows the spontaneous formation of multiple bimetallic catalytic sites capable to promote the cleavage of a RNA model substrate. We show that by tuning the structure of the nanoparticle-coating monolayer, it is possible to decrease the polarity of the reaction site, and this in turn generates remarkable increments of the cleavage efficiency.

Light-triggered thiol-exchange on gold nanoparticles at low micromolar concentrations in water.

The place-exchange reaction of thiol-containing peptides in a cationic monolayer on gold nanoparticles occurs very rapidly at low micromolar concentrations in water with excellent control over the degree of substitution. The driving force for this process is the binding of anionic peptides to a cationic monolayer surface which causes a strong increase in the local concentration of thiols. The place-exchange reaction can be triggered by light using a photolabile protecting group on the thiol moiety.

Zn2+-regulated self-sorting and mixing of phosphates and carboxylates on the surface of functionalized gold nanoparticles.

Herein, we describe the self-sorting of phosphate- and carboxylate-containing molecules on the surface of monolayer-protected gold nanoparticles. Self-sorting is driven by selective interactions between the phosphate probe and Zn(2+) complexes in one monolayer; these interactions force the carboxylate probe to move to a second type of nanoparticle. This process effectively separates the probes and causes their localization in well-defined spaces surrounding the nanoparticles. The removal/addition of Zn(2+) metal ions from the system is used to convert the system from an ordered to a disordered state and vice versa. The possibility to control the location and transport of populations of molecules in a complex mixture creates new perspectives for the development of innovative complex catalytic systems that mimic nature.

The Interaction of Amines with Gold Nanoparticles.

Understanding the interactions between amines and the surface of gold nanoparticles is important because of their role in the stabilization of the nanosystems, in the formation of the protein corona, and in the preparation of semisynthetic nanozymes. By using fluorescence spectroscopy, electrochemistry, X-ray photoelectron spectroscopy, high-resolution transmission electron microscopy, and molecular simulation, a detailed picture of these interactions is obtained. Herein, it is shown that amines interact with surface Au(0) atoms of the nanoparticles with their lone electron pair with a strength linearly correlating with their basicity corrected for steric hindrance. The kinetics of binding depends on the position of the gold atoms (flat surfaces or edges) while the mode of binding involves a single Au(0) with nitrogen sitting on top of it. A small fraction of surface Au(I) atoms, still present, is reduced by the amines yielding a much stronger Au(0)-RN.+ (RN. , after the loss of a proton) interaction. In this case, the mode of binding involves two Au(0) atoms with a bridging nitrogen placed between them. Stable Au nanoparticles, as those required for robust semisynthetic nanozymes preparation, are better obtained when the protein is involved (at least in part) in the reduction of the gold ions.

Catalysis of transesterification reactions by a self-assembled nanosystem.

Histidine-containing peptides self-assemble on the surface of monolayer protected gold nanoparticles to form a catalytic system for transesterification reactions. Self-assembly is a prerequisite for catalysis, since the isolated peptides do not display catalytic activity by themselves. A series of catalytic peptides and substrates are studied in order to understand the structural parameters that are of relevance to the catalytic efficiency of the system. It is shown that the distance between the His-residue and the anionic tail does not affect the catalytic activity. On the other hand, the catalytic His-residue is sensitive to the chemical nature of the flanking amino acid residues. In particular, the presence of polar Ser-residues causes a significant increase in activity. Finally, kinetic studies of a series of substrates reveal that substrates with a hydrophobic component are very suitable for this catalytic system.

Self-assembly of a catalytic multivalent peptide-nanoparticle complex.

Catalytically active peptide-nanoparticle complexes were obtained by assembling small peptide sequences on the surface of cationic self-assembled monolayers on gold nanoparticles. When bound to the surface, the peptides accelerate the transesterification of the p-nitrophenyl ester of N-carboxybenzylphenylalanine by more than 2 orders of magnitude. The gold nanoparticle serves as a multivalent scaffold for bringing the catalyst and substrate into close proximity but also creates a local microenvironment that further enhances the catalysis. The supramolecular nature of the ensemble permits the catalytic activity of the system to be modulated in situ.

Lanthanide-based NMR: a tool to investigate component distribution in mixed-monolayer-protected nanoparticles.

Gd(3+) ions, once bound to the monolayer of organic molecules coating the surface of gold nanoparticles, produce a paramagnetic relaxation enhancement (PRE) that broadens and eventually cancels the signals of the nuclear spins located nearby (within 1.6 nm distance). In the case of nanoparticles coated with mixed monolayers, the signals arising from the different coating molecules experience different PRE, depending on their distance from the binding site. As a consequence, observation of the signal broadening patterns provides direct information on the monolayer organization.