Tristhiolato Pseudopeptides Bind Arsenic(III) in an AsS3 Coordination Environment Imitating Metalloid Binding Sites in Proteins.

The AsIII binding of two NTA-based tripodal pseudopeptides, possessing three cysteine (ligand L1) or d-penicillamine residues (ligand L2) as potential coordinating groups for soft semimetals or metal ions, was studied by experimental (UV, CD, NMR, and ESI-MS) and theoretical (DFT) methods. All of the experimental data, obtained with the variation of the AsIII:ligand concentration ratios or pH values in some instances, evidence the exclusive formation of species with an AsS3-type coordination mode. The UV-monitored titration of the ligands with arsenous acid at pH = 7.0 provided an absorbance data set that allowed for the determination of apparent stability constants of the forming species. The obtained stabilities (logK' = 5.26 (AsL1) and logK' = 3.04 (AsL2)) reflect high affinities, especially for the sterically less restricted cysteine derivative. DFT calculated structures correlate well with the spectroscopic results and, in line with the 1H NMR data, indicate a preference for the all-endo conformers resembling the AsIII environment at the semimetal binding sites in various metalloproteins.

A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Mononuclear Ni(II) Complexes with a S3O Coordination Sphere Based on a Tripodal Cysteine-Rich Ligand: pH Tuning of the Superoxide Dismutase Activity.

The superoxide dismutase (SOD) activity of mononuclear NiII complexes, whose structures are inspired by the NiSOD, has been investigated. They have been designed with a sulfur-rich pseudopeptide ligand, derived from nitrilotriacetic acid (NTA), where the three acid functions are grafted with cysteines (L3S). Two mononuclear complexes, which exist in pH-dependent proportions, have been fully characterized by a combination of spectroscopic techniques including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations. They display similar square-planar S3O coordination, with the three thiolates of the three cysteine moieties from L3S coordinated to the NiII ion, together with either a water molecule at physiological pH, as [NiL3S(OH2)]-, or a hydroxo ion in more basic conditions, as [NiL3S(OH)]2-. The 1H NMR study has revealed that contrary to the hydroxo ligand, the bound water molecule is labile. The cyclic voltammogram of both complexes displays an irreversible one-electron oxidation process assigned to the NiII/NiIII redox system with Epa = 0.48 and 0.31 V versus SCE for NiL3S(OH2) and NiL3S(OH), respectively. The SOD activity of both complexes has been tested. On the basis of the xanthine oxidase assay, an IC50 of about 1 µM has been measured at pH 7.4, where NiL3S(OH2) is mainly present (93% of the NiII species), while the IC50 is larger than 100 µM at pH 9.6, where NiL3S(OH) is the major species (92% of the NiII species). Interestingly, only NiL3S(OH2) displays SOD activity, suggesting that the presence of a labile ligand is required. The SOD activity has been also evaluated under catalytic conditions at pH 7.75, where the ratio between NiL3S(OH2)/ NiL3S(OH) is about (86:14), and a rate constant, kcat = 1.8 × 105 M-1 s-1, has been measured. NiL3S(OH2) is thus the first low-molecular weight, synthetic, bioinspired Ni complex that displays catalytic SOD activity in water at physiological pH, although it does not contain any N-donor ligand in its first coordination sphere, as in the NiSOD. Overall, the data show that a key structural feature is the presence of a labile ligand in the coordination sphere of the NiII ion.

Quantification of Surface GalNAc Ligands Decorating Nanostructured Lipid Carriers by UPLC-ELSD.

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency.

A Trishistidine Pseudopeptide with Ability to Remove Both CuΙ and CuΙΙ from the Amyloid-ß Peptide and to Stop the Associated ROS Formation.

The pseudopeptide L, derived from a nitrilotriacetic acid scaffold and functionalized with three histidine moieties, is reminiscent of the amino acid side chains encountered in the Alzheimer's peptide (Aß). Its synthesis and coordination properties for CuΙ and CuΙΙ are described. L efficiently complex CuΙΙ in a square-planar geometry involving three imidazole nitrogen atoms and an amidate-Cu bond. By contrast, CuΙ is coordinated in a tetrahedral environment. The redox behavior is irreversible and follows an ECEC mechanism in accordance with the very different environments of the two redox states of the Cu center. This is in line with the observed resistance of the CuΙ complex to oxidation by oxygen and the CuΙΙ complex reduction by ascorbate. The affinities of L for CuΙΙ and CuΙ at physiological pH are larger than that reported for the Aß peptide. Therefore, due to its peculiar Cu coordination properties, the ligand L is able to target both redox states of Cu, redox silence them and prevent reactive oxygen species production by the CuAß complex. Because reactive oxygen species contribute to the oxidative stress, a key issue in Alzheimer's disease, this ligand thus represents a new strategy in the long route of finding molecular concepts for fighting Alzheimer's disease.

ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

Pseudo-peptides based on methyl cysteine or methionine inspired from Mets motifs found in the copper transporter Ctr1.

Most proteins involved in Cu homeostasis bind to intracellular Cu(I) in stable Cu(S-Cys)x environments, thanks to well-conserved cysteine-rich sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper acquisition, binds Cu(I) in Cu(S-Met)3 environments in conserved methionine-rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a nitrilotriacetic acid scaffold and functionalized with three amino acids bearing thioether side chains, either methyl cysteine in T(1) or methionine in T(2), were synthesized as mimics of the Mets sequences found in Ctr1. These two ligands were obtained with good overall yields from commercial amino acids and demonstrate efficient chelating ability for Cu(I). Only one species, the mononuclear [CuT(1,2)](+) complex, was evidenced by electrospray ionization-mass spectroscopy (ESI-MS) and the circular dichroism signature obtained for the most constrained CuT(1) complex having the shortest side chains showed reorganization of the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2-10.3. The CuT(1,2) complexes are significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport protein Ctr1 (log K ≈ 5-6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson's disease treatments.

XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites.

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination number, with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, respectively, comparable to the values reported for crystalline Ag(I)-thiolate compounds. The bioinspired Cu(I) chelator L(1) is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a reference compound for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the average Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, respectively, both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chemistry of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.

D-Penicillamine tripodal derivatives as efficient copper(I) chelators.

New tripodal metal-chelating agents derived from nitrilotriacetic acid (NTA) and extended by three unnatural amino acids D-penicillamine (D-Pen) are presented. D-Pen is actually the drug most extensively used to treat copper (Cu) overload in Wilson's disease and as such is a very attractive building block for the design of chelating agents. D-Pen is also a bulkier analogue of cysteine, with the ß-methylene hydrogen atoms replaced by larger methyl groups. The hindrance of the gem-dimethyl group close to the thiol functions is demonstrated to influence the speciation and stability of the metal complexes. The ligands L(4) (ester) and L(5) (amide) were obtained from NTA and commercial D-Pen synthons in four and five steps with overall yields of 14 and 24%, respectively. Their ability to bind Cu(I), thanks to their three thiolate functions, has been investigated using both spectroscopic and analytical methods. UV, CD, and NMR spectroscopy and mass spectrometry evidence the formation of two Cu(I) complexes with L(5): the mononuclear complex CuL(5) and one cluster (Cu2L(5))2. In contrast, the bulkier ethyl ester derivative L(4) cannot accommodate the mononuclear complex in solution and thus forms exclusively the cluster (Cu2L(4))2. Cu K-edge X-ray absorption spectroscopy (XAS and EXAFS) confirms that Cu(I) is bound in trigonal-planar sulfur-only environments in all of these complexes with Cu- - -S distances ranging from 2.22 to 2.23 Å. Such C3-symmetric CuS3 cores are coordination modes frequently adopted in Cu(I) proteins such as metallothioneins. These two ligands bind Cu(I) tightly and selectively, which makes them promising chelators for intracellular copper detoxification in vivo.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

X-ray absorption spectroscopy proves the trigonal-planar sulfur-only coordination of copper(I) with high-affinity tripodal pseudopeptides.

A series of tripodal ligands L derived from nitrilotriacetic acid (NTA) and extended by three converging metal-binding cysteine chains were previously found to bind selectively copper(I) both in vitro and in vivo. The ligands L(1) (ester) and L(2) (amide) were demonstrated to form copper(I) species with very high affinities, close to that reported for the metal-sequestering metallothioneins (MTs; log K(Cu-MT) ≈ 19). Here, an in-depth study by Cu K-edge X-ray absorption spectroscopy (XAS) was performed to completely characterize the copper(I) coordination sphere in the complexes, previously evidenced by other physicochemical analyses. The X-ray absorption near-edge structure (XANES) spectra shed light on the equilibrium between a mononuclear complex and a cluster for both L(1) (ester) and L(2) (amide). The exclusive symmetric CuS3 geometry adopted in the mononuclear complexes (Cu-S ≈ 2.23 Å) was clearly demonstrated by extended X-ray absorption fine structure (EXAFS) analyses. The EXAFS analyses also proved that the clusters are organized on a symmetric CuS3 core (Cu-S ≈ 2.26 Å) and interact with three nearby copper atoms (Cu---Cu ≈ 2.7 Å), consistent with the Cu6S9-type clusters previously characterized by pulsed gradient spin echo NMR spectroscopy. XAS data obtained for other architectures based on the NTA template (L(3) acid, L(4) without a functionalized carbonyl group, etc.) demonstrated the formation of polymetallic species only, which evidence the necessity of the proximal ester or amide group to stabilize the CuS3 mononuclear species. Finally, XAS was demonstrated to be a powerful method to quantify the equilibrium between the two copper(I) environments evidenced with L(1) and L(2) at different copper concentrations and to determine the equilibrium constants between these two complexes.

Metal-controlled diastereoselective self-assembly and circularly polarized luminescence of a chiral heptanuclear europium wheel.

The chiral dissymmetric tetradentate ligand (S)-6'-(4-phenyloxazolin-2-yl)-2,2'-bipyridine-6-carboxylate (S-Phbipox) leads to the diastereoselective assembly of a homochiral Eu(3+) triangle and a highly emissive (quantum yield = 27%) heptanuclear wheel that is the largest example of a chiral luminescent complex of Eu(3+) reported to date. The nuclearity of the assembly is controlled by the solvent and the Eu(3+) cation. All of the compounds show large circularly polarized luminescence with an activity that varies with the nature of the assembly (highest for the homochiral trimer).

Hepatocyte targeting and intracellular copper chelation by a thiol-containing glycocyclopeptide.

Metal overload plays an important role in several diseases or intoxications, like in Wilson's disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.

A series of tripodal cysteine derivatives as water-soluble chelators that are highly selective for copper(I).

A series of tripodal ligands derived from nitrilotriacetic acid and extended by three converging, metal-binding, cysteine chains was synthesised. Their ability to bind soft metal ions thanks to their three thiolate functions was investigated by means of complementary analytical and spectroscopic methods. Three ligands that differ by the nature of the carbonyl group next to the coordinating thiolate functions were studied: L(1) (ester), L(2) (amide) and L(3) (carboxylate). The negatively charged derivative L(3), which bears three carboxylate functions close to the metal binding site, gives polynuclear copper(I) complexes of low stability. In contrast, the ester and amide derivatives L(1) and L(2) are efficient Cu(I) chelators with very high affinities, close to that reported for the metal-sequestering metallothioneins (log K≈19). Interestingly, these two ligands form mononuclear copper complexes with a unique MS(3) coordination in water solution. An intramolecular hydrogen-bond network involving the amide functions in the upper cavity of the tripodal ligands stabilises these mononuclear complexes and was evidenced by the very low chemical-shift temperature coefficient of the secondary amide protons. Moreover, L(1) and L(2) display large selectivities for the targeted metal ion that is, Cu(I), with respect to bioavailable Zn(II). Therefore the two sulfur-based tripods L(1) and L(2) are of potential interest for intracellular copper detoxication in vivo, without altering the homeostasis of the essential metal ion Zn(II).

Development, formulation, and cellular mechanism of a lipophilic copper chelator for the treatment of Wilson's disease.

Copper homeostasis is finely regulated in human to avoid any detrimental impact of free intracellular copper ions. Upon copper accumulation, biliary excretion is triggered in liver thanks to trafficking of the ATP7B copper transporter to bile canaliculi. However, in Wilson's disease this protein is mutated leading to copper accumulation. Current therapy uses Cu chelators acting extracellularly and requiring a life-long treatment with side effects. Herein, a new Cu(I) pro-chelator was encapsulated in long-term stable nanostructured lipid carriers. Cellular assays revealed that the pro-chelator protects hepatocytes against Cu-induced cell death. Besides, the cellular stresses induced by moderate copper concentrations, including protein unfolding, are counteracted by the pro-chelator. These data showed the pro-chelator efficiency to deliver intracellularly an active chelator that copes with copper stress and surpasses current and under development chelators. Although its biological activity is more mitigated, the pro-chelator nanolipid formulation led to promising results. This innovative approach is of outmost importance in the quest of better treatments for Wilson's disease.

Tripodal scaffolds with three appended imidazole thiones for Cu(I) chelation and protection from Cu-mediated oxidative stress.

Imidazole thiones appear as interesting building blocks for Cu(I) chelation and protection against Cu-mediated oxidative stress. Therefore, a series of tripodal molecules derived from nitrilotriacetic acid appended with three imidazole thiones belonging either to histamine-like or histidine-like moieties were synthesized. These tripods demonstrate intermediate affinity between that previously measured for tripodal analogues bearing three thiol moieties such as cysteine and those grafted with three thioethers, like methionines, consistently with the thione group in the imidazole thione moiety existing as a tautomer between a thiol and a thione. The two non-alkylated tripods derived from thioimidazole, TH and TH* demonstrated three orders of magnitude larger affinity for Cu(I) (logKpH 7.4 = 14.3) than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt (logKpH 7.4 = 11-11.6). Their efficiency to inhibit Cu-mediated oxidative stress is demonstrated by several assays involving ascorbate consumption or biomolecule damages and correlates with their ability to chelate Cu(I), related to their conditional complexation constants at pH 7.4. The two non-alkylated tripods derived from thioimidazole, TH and TH* are significantly more powerful in reducing Cu-mediated oxidative stress than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

A liver-targeting Cu(i) chelator relocates Cu in hepatocytes and promotes Cu excretion in a murine model of Wilson's disease.

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson's disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson's disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.

In vitro assessment of cobalt oxide particle dissolution in simulated lung fluids for identification of new decorporating agents.

Inhalation of 60Co3O4 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to γ rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1-28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance 60Co3O4 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.

Safer-by-design biocides made of tri-thiol bridged silver nanoparticle assemblies.

Silver nanoparticles (AgNPs) are efficient biocides increasingly used in consumer products and medical devices. Their activity is due to their capacity to release bioavailable Ag(i) ions making them long-lasting biocides but AgNPs themselves are usually easily released from the product. Besides, AgNPs are highly sensitive to various chemical environments that triggers their transformation, decreasing their activity. Altogether, widespread use of AgNPs leads to bacterial resistance and safety concerns for humans and the environment. There is thus a crucial need for improvement. Herein, a proof of concept for a novel biocide based on AgNP assemblies bridged together by a tri-thiol bioinspired ligand is presented. The final nanomaterial is stable and less sensitive to chemical environments with AgNPs completely covered by organic molecules tightly bound via their thiol functions. Therefore, these AgNP assemblies can be considered as safer-by-design and innovative biocides, since they deliver a sufficient amount of Ag(i) for biocidal activity with no release of AgNPs, which are insensitive to transformations in the nanomaterial.