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Open questions on proteins interacting with nanoclusters

Open questions on proteins interacting with nanoclusters COMMENT https://doi.org/10.1038/s42004-022-00665-9 OPEN Open questions on proteins interacting with nanoclusters 1 2 3 Rodolphe Antoine , Dusica Maysinger , Lucie Sancey & 4,5,6 Vlasta Bonačić-Koutecký Interfacing ultrasmall metal nanoclusters (NCs) with proteins can present a dual opportunity: proteins can be used for protecting NCs, and the surface ligands of NCs may interact with proteins. Here, the authors identify and discuss remaining open questions surrounding the bio-NC interface that call for future research efforts. Ligand protected metal nanoclusters (NCs) have attracted increasing attention due to their fascinating physicochemical properties, in particular luminescence , and their capability to be functional for biomedical applications. Such capability can be achieved by interfacing NCs with biomolecules, in particular proteins (bio-NC interface, Fig. 1a). The bio-NC interface presents a dual role (Fig. 1b, c): on the one hand, proteins can be used for protecting NCs, and on the other hand, surface ligands of NCs may present some interaction (intended or not) with proteins. Here we focus on open questions arising from the interaction of NCs with proteins, considering in vitro and in vivo scenarios and using in silico approaches. In particular, we explore what are the main problems associated with nanocluster functionalization by proteins and what happens within exchange reactions of these proteins with intracellular and extracellular endogenous proteins in living cells? How robust is the bio-NC interface? Could nanoclusters qualify as alternatives or substitutes for some fluorescent dyes in biological sciences? Proteins interacting with ultrasmall metal NCs As bare metal clusters are not stable in solution, they need to be protected. The use of proteins as a protective template for metal NC synthesis and functionalization is a rather elegant strategy 2,3 used in the NC community. The marriage of metal NCs with biomolecules , such as proteins, may create some synergistic effects combining the unique optical, electronic, and catalytic 3,4 properties of the metal NCs with the biological functions of proteins . A pioneering work in the field of bio-NC interface was reported by Xie et al. describing an elegant and simple one-pot 4,5 aqueous synthesis of fluorescent bovine serum albumin–gold (BSA–Au) NCs . Following up on this seminal work, a plethora of NCs were synthesized with different proteins for biomedical 4,5 purposes . However, due to the fact that such one-pot synthesis uses proteins both as reductant and stabilizer agents, some alteration of the structure of the protein host and/or uncontrolled formation of clusters in uncontrolled locations inside the protein occurs , limiting the wide- spread use of such materials in biomedical research. Therefore, new synthetic methods preser- ving the bioactivity of the protein-directed NCs as well as the structure of the incorporated atomically precise metal cluster is greatly needed. Such synthetic routes could be obtained using 1 2 ̀ ̀ Institut Lumiere Matiere UMR 5306, Université Claude Bernard Lyon 1, CNRS, Univ Lyon, F- 69100 Villeurbanne, France. Department of Pharmacology & Therapeutics, McGill University, Montréal, QC H3G 1Y6, Canada. Cancer Targets and Experimental Therapeutics, Institute for Advanced Biosciences (IAB), INSERM-U1209/CNRS-UMR 5309, University of Grenoble Alpes (UGA), 38000 Grenoble, France. Center of Excellence for Science and Technology- Integration of Mediterranean Region (STIM), Faculty of Science, University of Split, 21000 Split, Croatia. Interdisciplinary Center for Advanced Science and Technology (ICAST) at University of Split, 21000 Split, Croatia. Chemistry Department, Humboldt University of Berlin, 12489 Berlin, Germany. email: rodolphe.antoine@univ-lyon1.fr; vbk@cms.hu-berlin.de COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem 1 1234567890():,; COMMENT COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 Fig. 1 Dual opportunity of the bio-NC interface: proteins can be used for protecting NCs, and the surface ligands of NCs may interact with proteins. a Schematics of ligand protected metal NCs and proteins leading to the bio-NC interface. Dual role of the bio-NC interface: b proteins can be used as templates for protecting NCs and c surface ligands of NCs may be functionalized to present some specific interactions with proteins. ligand exchange to attach a protein molecule onto preformed Therefore, the toxicity of NCs both in vitro and in vivo must be atomically precise metal NCs. This specific binding was studied assessed before any clinical application should be considered. mechanistically using a computational approach and demon- Despite numerous biological applications of NCs in recent years, strated experimentally as well as supported theoretically very knowledge of their interactions within the complex biological recently by our groups , where a preformed Au NC was environment, in particular the cellular medium, is still limited. incorporated in bovine serum albumin (BSA) protein by ligand For instance, it is still unclear how the surface interactions exchange between the NC surface ligands and BSA cysteine between cellular proteins and NCs affect the biological activity. residues (Fig. 1b). This strategy could be further advanced by The uptake mechanisms of NCs also remain unexplored in a engineering mutations in proteins with cysteines, allowing for systematic manner. Considering that different cell types are more selective control of the position of atomically precise metal equipped with different surface proteins and membrane proper- NCs in proteins and of the number of ligands exchanged. ties and that they exist in different states (e.g., physiological, Exploring the dual role of the bio-NC interface will also consist pathological, activated, stimulated, etc.), and considering the in using “click chemistry” approaches to attach a protein molecule complexity of ligand arrangements on nanoclusters of different onto an atomically precise metal NC that has well-characterized shapes and sizes, systematic study of uptake mechanisms is an structural and optical properties. For example, using highly enormous and difficult task. Although many reports have indi- luminescent metal NCs, a protecting ligand could be exchanged cated that some NCs have good biocompatibility, a thorough for a protein molecule using a targeting linker specifically study of the key ingredients affecting the in vitro toxicity merits designed. We developed such a strategy to enhance the detection further investigation. A comprehensive examination of survival of protein carbonyls by optical methods. Glutathione (SG)-pro- and cell death mechanisms needs to be made, particularly in tected gold NCs (Au SG ) were readily functionalized via thio- human cells. We recently demonstrated that ligand protected gold 15 13 lated aminooxy by a ligand exchange procedure. The as-prepared (Au) NCs were not inert in human primary astrocytes but functionalized Aminooxy-Au NCs reacted with carbonylated revealed organellar reorganization upon AuNC treatment, as well proteins (see Fig. 1c). The targeted carbonylated proteins were as the activation of key cytoprotective transcription factors. Size 9 12 then detected by either one- or two-photon fluorescence , and and ligand effects are definitely playing a key role . Once again, such functionalized NCs could be a useful approach for cellular such an effect might be related to the capability of AuNCs to react imaging applications . Other targeting linkers could be coupled with proteins through ligand exchange reactions. Also, similar to through azide-alkyne cycloaddition reactions . The incorporation larger AuNCs, intracellular reactive oxygen species (ROS) gen- of a reactive azide group presents challenges for developing eration by NCs was one of the important contributors to 13,14 functional NCs for applications in glycobiology and analysis of the mechanisms of toxicity . However, the advantage of pro- carbohydrate-protein interaction. ducing nanoclusters with atomic precision allows for unam- biguously establishing the links connecting the structure of nanoclusters with their properties upon changes in several bio- Open questions on proteins interacting with NCs in vitro molecules under oxidative stress, as nicely exemplified with Nowadays, contradictory results exist for cytotoxicity properties Au SG (see Fig. 2a). Some of the undesirable effects could be of metal NCs. Many factors such as different experimental 10 10 alleviated, at least in part, by scavengers of the ROS and by an designs, size and ligands used, different types of tumor cells or increase in the protective ligand stability. untransformed primary cultures may explain such inconsistency. 2 COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 COMMENT Fig. 2 proteins interacting with NCs in vitro and vivo. a Illustration of the impact of gold nanoclusters Au SG on human microglia. Different endpoint 10 10 parameters used to determine Au SG toxicity in microglial cells. Reduced form of HMGB1 (redHMGB1: gray ribbon representation for the A box domain 10 10 of redHMGB1 and the beige ribbon for the B box domain) interacts with glutathione ligands at the surface of Au SG (stick representation). b Au NCs 10 10 25 attached to BSA (illustrated in Fig. 1b) present impressive luminescence properties in SWIR (i.e., emission 1064–1700 nm) enabling in vivo imaging, but extremely short residence time (serum half-life = 3.6 min) and very low cancer targeting potential as displayed with bio-distribution and pharmacokinetics investigations. Error bars represent standard deviations of measurements. Open questions on proteins interacting with NCs in vivo Open questions on in silico approaches for proteins Concerning the practical use of NCs in clinical applications in interacting with NCs particular cancer theranostics, there is often a trade-off between As mentioned, the production of nanoclusters with atomic preci- 15,16 residence time in the circulation and cellular uptake . Once sion allows for unambiguously establishing the links connecting the again, size and ligand influence affect the biodistribution, tar- structure of nanoclusters with their properties upon changes in geting potential, renal elimination, tumor uptake, and biode- several biomolecular environments. Advances in computational gradability of metal NCs. For instance, preformed thiolated chemistry and hybrid calculations of large systems, e.g., with protected Au NCs attached to BSA present impressive lumi- quantum mechanical (density functional)/molecular mechanical nescence properties in short-wave infrared enabling in vivo QM/MM) treatment for systems containing one thousand atoms, imaging (i.e., >1000 nm), but extremely short residence time and should also make an impact on understanding the most probable very low cancer targeting potential, as commonly observed with binding sites for NCs within a protein molecule and the effect of 15,16 gold nanoclusters (Fig. 2b) . Each of these parameters must be protein residues in controlling NC size and structure (in silico carefully addressed when designing NCs for specific biomedical approaches) . Such hybrid methods can address photophysical applications and their possible clinical translation. There is still processes (luminescence in the linear and nonlinear optical regime) room for improving their performance and fate in human tissue, in protein-NC systems. A combined experimental and theoretical including their circulation in blood and clearance from the body. approach including computational simulation tools can provide a In particular, we showed that linking AuNCs through the ligand holistic description of the nature of the interactions present at the exchange strategy within protein templates can have a strong protein-NC interface. This will allow for the establishment of rules enhancement effect on their linear and nonlinear optical lumi- for predicting optimal protein-NC interaction in order to initiate nescence properties in the NIR-II region . Longer circulation new experiments focused on applications. times and deeper accumulation within the tumor might be Theaim whichshouldberealizedinthe future is theoptimization achieved by post-functionalization strategies, and ligand pro- of the efficiency of protein- NC interactions in the context of tected metal NCs were found to be ideal templates. One of the applications. Present achievements are based on general character- main advantages of ligand protected NCs is the possibility of izations of ligated noble metal nanoclusters representing the class of creating a NC platform with functional moieties appended onto ultrasmall nanomaterials with distinctive structural and optical each surface ligand, dramatically enhancing targeting sensitivity properties, which have yet to be extended beyond the proof of toward cancer materials. principle. This means that the optimal size of NCs and the choice of COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem 3 COMMENT COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 ligandshavetobedesignedfor efficient interaction with proteins in 4. Xie, J., Zheng, Y. & Ying, J. Y. Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 131, 888–889 (2009). the context of improving desirable optical properties, such as high 5. Zare, I. et al. Protein-protected metal nanoclusters as diagnostic and brightness and photo-stability needed for numerous applications in therapeutic platforms for biomedical applications. Materials Today https:// medical diagnostics and medical treatments. The ligands play an doi.org/10.1016/j.mattod.2020.10.027 (2021). additional key role since they participate in drastically enhancing 6. Soleilhac, A., Bertorelle, F. & Antoine, R. Sizing protein-templated gold non-linear optical properties (NLO) of liganded NCs in the NIR nanoclusters by time resolved fluorescence anisotropy decay measurements. Spectrochimica Acta Part A Mol. Biomolecular Spectrosc. 193, 283–288 (2018). regime . Moreover, functionalization can be achieved by ligand 7. Rojas-Cervellera, V., Raich, L., Akola, J. & Rovira, C. The molecular exchange. An example is functionalized aminooxy Au SG NCs 15 13 mechanism of the ligand exchange reaction of an antibody against a (obtained by ligand exchange) interacting with carbonylated proteins. glutathione-coated gold cluster. Nanoscale 9, 3121–3127 (2017). Protein carbonylation at the molecular level on model lysozyme was 8. Bertorelle, F. et al. Tailoring NIR-II photoluminescence of single thiolated investigated by computational chemistry to determine the nature of Au25 nanoclusters by selective binding to proteins. Research Square https:// doi.org/10.21203/rs.3.rs-958149/v1 (2022). bindingbetween theNCs andthe proteincarbonyls (see Fig. 1c). 9. Combes, G. F. et al. Functionalized Au15 nanoclusters as luminescent probes Altogether, this is proof of principle that functionalized liganded for protein carbonylation detection. Commun Chem. 4, 69 (2021). AuNCs can serve for the detection of carbonylation sites and might 10. Bonačić-Koutecký, V. & Antoine, R. Enhanced two-photon absorption of be more efficient than organic dyes. Such rational design of novel ligated silver and gold nanoclusters: theoretical and experimental assessments. functional thiolate-protected metal NCs with controllable surface Nanoscale 11, 12436–12448 (2019). 11. Gunawardene, P. N., Corrigan, J. F. & Workentin, M. S. Golden opportunity: a chemistry could open new avenues towards many other practical clickable azide-functionalized [Au25(SR)18]− nanocluster platform for applications, particularly in bioimaging and early medical diagnostics. interfacial surface modifications. J. Am. Chem. Soc. 141, 11781–11785 (2019). 12. Gran, E. R. et al. Size and ligand effects of gold nanoclusters in alteration of Outlook organellar state and translocation of transcription factors in human primary astrocytes. Nanoscale 13, 3173–3183 (2021). The recent studies mentioned above show both the possibility and 13. Dong, L. et al. Cytotoxicity of BSA-stabilized gold nanoclusters: in vitro and the need to further investigate the nature and the stability of the bio- in vivo study. Small 11, 2571–2581 (2015). NC interface. Advances in electron/X-ray techniques for structural 14. Maysinger, D. et al. Insights into the impact of gold nanoclusters Au10SG10 characterization are absolutely mandatory to better understand the on human microglia. ACS Chem. Neurosci. 13, 464–476 (2022). protein-NC interactions in complex biological media, but also to 15. Colombé, C. et al. Gold nanoclusters as a contrast agent for image-guided surgery of head and neck tumors. Nanomed. Nanotechnol., Biol. Med. 20, 102011 (2019). probe structural and chemical changes that occur in situ and in real 16. Shen, D. et al. Zwitterion functionalized gold nanoclusters for multimodal near time. Mass spectrometry should also bridge the gap to characterizing infrared fluorescence and photoacoustic imaging. APL Mater. 5, 053404 (2017). increasingly complex ligated metal nanoclusters (using zwitterionic 17. Perić, M. et al. Ligand shell size effects on one- and two-photon excitation ligands, proteins, multi-shell ligands) .Advancementsinin situ fluorescence of zwitterion functionalized gold nanoclusters. Phys. Chem. spectroscopic measurements will lead to a better understanding of Chem. Phys. 21, 23916–23921 (2019). 18. Antoine R. Bonacic-Koutecky V. Liganded silver and gold quantum clusters. how protein-NCs perform in biological environments and conditions Towards a new class of nonlinear optical nanomaterials. SpringerBriefs in and will help towards improved designs for their intended applica- Materials. (Springer, Cham, 2018). https://doi.org/10.1007/978-3-319-64743-2_1. tions, including the reinforcement of the link between the AuNC and 19. Comby-Zerbino, C., Dagany, X., Chirot, F., Dugourd, P. & Antoine, R. The the protein for a longer biological stability. emergence of mass spectrometry for characterizing nanomaterials. Atomically Super resolution microscopy with life-time imaging and precise nanoclusters and beyond. Mater. Adv. 2, 4896–4913 (2021). advanced electron microscopy methods allowing for 3D recon- structions from living cells will add to our still limited knowledge of Acknowledgements nanocluster fate in diverse cell types and their physiological and The idea for this manuscript came up during the participation of all four co-authors at pathological conditions. Kinetic parameters showing residence time the Research Workshop “Interdisciplinary Endeavour in Technology at Nanoscale, Water and Environment” (9–11 October 2019), MedILS, Split, Croatia, organized by the of nanoclusters in different organelles are currently not available. head, prof. dr. dr. h. c. Vlasta Bonačić-Koutecký, of the Center of Excellence for Science These would require challenging experimental protocols but they and Technology - Integration of Mediterranean Region, Research, Innovation, Education, could provide much needed information leading to a better idea (STIM-REI) at the University of Split, Croatia. (Contract Number: KK.01.1.1.01.0003, a about organelle-associated proteins and their roles in interacting project funded by the European Union through the European Regional Development with nanoclusters. Human cell organoids are valuable model sys- Fund – the Operational Programme Competitiveness and Cohesion 2014–2020 (KK.01.1.1.01)). The contribution within the activity “Design of novel biosensing tems to explore nanoclusters in a more complex cellular environ- materials and their application in medical diagnostics in biomedicine” extended the ment, complementing those in monolayer cultures and co-cultures. existing theoretical and experimental network (Berlin- Split- Lyon), opening and addi- Computational contributions in establishing rules for designing tional interdisciplinary avenue in medical application in vitro (Montreal) and in vivo appropriate interactions between proteins and nanoclusters can (Grenoble). These research directions resulted in a novel approach towards nanocluster- contribute to motivate new appropriate experiments. The joint bio interaction with proteins. As a chain reaction in science this approach opens new questions to be answered in the future. The co-authors would like to express their efforts of physical and theoretical chemists, material scientists and gratitude to the collaborators: Franck Bertorelle, Martina Perić Bakulić, Željka Sanader biologists are expected to propel protein-protected NCs to ever- Maršić and Evan Rizel Gran. greater achievements in biological applications. These joint efforts will contribute in the future to the further development of new Author contributions nanoclusters with optimized properties. R.A., D.M., L.S. and V.B.-K. contributed equally to the manuscript. Received: 19 February 2022; Accepted: 14 March 2022; Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to Rodolphe Antoine or References Vlasta Bonačić-Koutecký. 1. Kang, X. & Zhu, M. Tailoring the photoluminescence of atomically precise Reprints and permission information is available at http://www.nature.com/reprints nanoclusters. Chem. Soc. Rev. 48, 2422–2457 (2019). 2. Tabarin, T. et al. Absorption enhancement and conformational control of peptides by small silver clusters. Phys. Rev. Lett. 101, 213001 (2008). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 3. Goswami, N., Zheng, K. & Xie, J. Bio-NCs – the marriage of ultrasmall metal published maps and institutional affiliations. nanoclusters with biomolecules. Nanoscale 6, 13328–13347 (2014). 4 COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 COMMENT Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2022 COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem 5 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Communications Chemistry Springer Journals

Open questions on proteins interacting with nanoclusters

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COMMENT https://doi.org/10.1038/s42004-022-00665-9 OPEN Open questions on proteins interacting with nanoclusters 1 2 3 Rodolphe Antoine , Dusica Maysinger , Lucie Sancey & 4,5,6 Vlasta Bonačić-Koutecký Interfacing ultrasmall metal nanoclusters (NCs) with proteins can present a dual opportunity: proteins can be used for protecting NCs, and the surface ligands of NCs may interact with proteins. Here, the authors identify and discuss remaining open questions surrounding the bio-NC interface that call for future research efforts. Ligand protected metal nanoclusters (NCs) have attracted increasing attention due to their fascinating physicochemical properties, in particular luminescence , and their capability to be functional for biomedical applications. Such capability can be achieved by interfacing NCs with biomolecules, in particular proteins (bio-NC interface, Fig. 1a). The bio-NC interface presents a dual role (Fig. 1b, c): on the one hand, proteins can be used for protecting NCs, and on the other hand, surface ligands of NCs may present some interaction (intended or not) with proteins. Here we focus on open questions arising from the interaction of NCs with proteins, considering in vitro and in vivo scenarios and using in silico approaches. In particular, we explore what are the main problems associated with nanocluster functionalization by proteins and what happens within exchange reactions of these proteins with intracellular and extracellular endogenous proteins in living cells? How robust is the bio-NC interface? Could nanoclusters qualify as alternatives or substitutes for some fluorescent dyes in biological sciences? Proteins interacting with ultrasmall metal NCs As bare metal clusters are not stable in solution, they need to be protected. The use of proteins as a protective template for metal NC synthesis and functionalization is a rather elegant strategy 2,3 used in the NC community. The marriage of metal NCs with biomolecules , such as proteins, may create some synergistic effects combining the unique optical, electronic, and catalytic 3,4 properties of the metal NCs with the biological functions of proteins . A pioneering work in the field of bio-NC interface was reported by Xie et al. describing an elegant and simple one-pot 4,5 aqueous synthesis of fluorescent bovine serum albumin–gold (BSA–Au) NCs . Following up on this seminal work, a plethora of NCs were synthesized with different proteins for biomedical 4,5 purposes . However, due to the fact that such one-pot synthesis uses proteins both as reductant and stabilizer agents, some alteration of the structure of the protein host and/or uncontrolled formation of clusters in uncontrolled locations inside the protein occurs , limiting the wide- spread use of such materials in biomedical research. Therefore, new synthetic methods preser- ving the bioactivity of the protein-directed NCs as well as the structure of the incorporated atomically precise metal cluster is greatly needed. Such synthetic routes could be obtained using 1 2 ̀ ̀ Institut Lumiere Matiere UMR 5306, Université Claude Bernard Lyon 1, CNRS, Univ Lyon, F- 69100 Villeurbanne, France. Department of Pharmacology & Therapeutics, McGill University, Montréal, QC H3G 1Y6, Canada. Cancer Targets and Experimental Therapeutics, Institute for Advanced Biosciences (IAB), INSERM-U1209/CNRS-UMR 5309, University of Grenoble Alpes (UGA), 38000 Grenoble, France. Center of Excellence for Science and Technology- Integration of Mediterranean Region (STIM), Faculty of Science, University of Split, 21000 Split, Croatia. Interdisciplinary Center for Advanced Science and Technology (ICAST) at University of Split, 21000 Split, Croatia. Chemistry Department, Humboldt University of Berlin, 12489 Berlin, Germany. email: rodolphe.antoine@univ-lyon1.fr; vbk@cms.hu-berlin.de COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem 1 1234567890():,; COMMENT COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 Fig. 1 Dual opportunity of the bio-NC interface: proteins can be used for protecting NCs, and the surface ligands of NCs may interact with proteins. a Schematics of ligand protected metal NCs and proteins leading to the bio-NC interface. Dual role of the bio-NC interface: b proteins can be used as templates for protecting NCs and c surface ligands of NCs may be functionalized to present some specific interactions with proteins. ligand exchange to attach a protein molecule onto preformed Therefore, the toxicity of NCs both in vitro and in vivo must be atomically precise metal NCs. This specific binding was studied assessed before any clinical application should be considered. mechanistically using a computational approach and demon- Despite numerous biological applications of NCs in recent years, strated experimentally as well as supported theoretically very knowledge of their interactions within the complex biological recently by our groups , where a preformed Au NC was environment, in particular the cellular medium, is still limited. incorporated in bovine serum albumin (BSA) protein by ligand For instance, it is still unclear how the surface interactions exchange between the NC surface ligands and BSA cysteine between cellular proteins and NCs affect the biological activity. residues (Fig. 1b). This strategy could be further advanced by The uptake mechanisms of NCs also remain unexplored in a engineering mutations in proteins with cysteines, allowing for systematic manner. Considering that different cell types are more selective control of the position of atomically precise metal equipped with different surface proteins and membrane proper- NCs in proteins and of the number of ligands exchanged. ties and that they exist in different states (e.g., physiological, Exploring the dual role of the bio-NC interface will also consist pathological, activated, stimulated, etc.), and considering the in using “click chemistry” approaches to attach a protein molecule complexity of ligand arrangements on nanoclusters of different onto an atomically precise metal NC that has well-characterized shapes and sizes, systematic study of uptake mechanisms is an structural and optical properties. For example, using highly enormous and difficult task. Although many reports have indi- luminescent metal NCs, a protecting ligand could be exchanged cated that some NCs have good biocompatibility, a thorough for a protein molecule using a targeting linker specifically study of the key ingredients affecting the in vitro toxicity merits designed. We developed such a strategy to enhance the detection further investigation. A comprehensive examination of survival of protein carbonyls by optical methods. Glutathione (SG)-pro- and cell death mechanisms needs to be made, particularly in tected gold NCs (Au SG ) were readily functionalized via thio- human cells. We recently demonstrated that ligand protected gold 15 13 lated aminooxy by a ligand exchange procedure. The as-prepared (Au) NCs were not inert in human primary astrocytes but functionalized Aminooxy-Au NCs reacted with carbonylated revealed organellar reorganization upon AuNC treatment, as well proteins (see Fig. 1c). The targeted carbonylated proteins were as the activation of key cytoprotective transcription factors. Size 9 12 then detected by either one- or two-photon fluorescence , and and ligand effects are definitely playing a key role . Once again, such functionalized NCs could be a useful approach for cellular such an effect might be related to the capability of AuNCs to react imaging applications . Other targeting linkers could be coupled with proteins through ligand exchange reactions. Also, similar to through azide-alkyne cycloaddition reactions . The incorporation larger AuNCs, intracellular reactive oxygen species (ROS) gen- of a reactive azide group presents challenges for developing eration by NCs was one of the important contributors to 13,14 functional NCs for applications in glycobiology and analysis of the mechanisms of toxicity . However, the advantage of pro- carbohydrate-protein interaction. ducing nanoclusters with atomic precision allows for unam- biguously establishing the links connecting the structure of nanoclusters with their properties upon changes in several bio- Open questions on proteins interacting with NCs in vitro molecules under oxidative stress, as nicely exemplified with Nowadays, contradictory results exist for cytotoxicity properties Au SG (see Fig. 2a). Some of the undesirable effects could be of metal NCs. Many factors such as different experimental 10 10 alleviated, at least in part, by scavengers of the ROS and by an designs, size and ligands used, different types of tumor cells or increase in the protective ligand stability. untransformed primary cultures may explain such inconsistency. 2 COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 COMMENT Fig. 2 proteins interacting with NCs in vitro and vivo. a Illustration of the impact of gold nanoclusters Au SG on human microglia. Different endpoint 10 10 parameters used to determine Au SG toxicity in microglial cells. Reduced form of HMGB1 (redHMGB1: gray ribbon representation for the A box domain 10 10 of redHMGB1 and the beige ribbon for the B box domain) interacts with glutathione ligands at the surface of Au SG (stick representation). b Au NCs 10 10 25 attached to BSA (illustrated in Fig. 1b) present impressive luminescence properties in SWIR (i.e., emission 1064–1700 nm) enabling in vivo imaging, but extremely short residence time (serum half-life = 3.6 min) and very low cancer targeting potential as displayed with bio-distribution and pharmacokinetics investigations. Error bars represent standard deviations of measurements. Open questions on proteins interacting with NCs in vivo Open questions on in silico approaches for proteins Concerning the practical use of NCs in clinical applications in interacting with NCs particular cancer theranostics, there is often a trade-off between As mentioned, the production of nanoclusters with atomic preci- 15,16 residence time in the circulation and cellular uptake . Once sion allows for unambiguously establishing the links connecting the again, size and ligand influence affect the biodistribution, tar- structure of nanoclusters with their properties upon changes in geting potential, renal elimination, tumor uptake, and biode- several biomolecular environments. Advances in computational gradability of metal NCs. For instance, preformed thiolated chemistry and hybrid calculations of large systems, e.g., with protected Au NCs attached to BSA present impressive lumi- quantum mechanical (density functional)/molecular mechanical nescence properties in short-wave infrared enabling in vivo QM/MM) treatment for systems containing one thousand atoms, imaging (i.e., >1000 nm), but extremely short residence time and should also make an impact on understanding the most probable very low cancer targeting potential, as commonly observed with binding sites for NCs within a protein molecule and the effect of 15,16 gold nanoclusters (Fig. 2b) . Each of these parameters must be protein residues in controlling NC size and structure (in silico carefully addressed when designing NCs for specific biomedical approaches) . Such hybrid methods can address photophysical applications and their possible clinical translation. There is still processes (luminescence in the linear and nonlinear optical regime) room for improving their performance and fate in human tissue, in protein-NC systems. A combined experimental and theoretical including their circulation in blood and clearance from the body. approach including computational simulation tools can provide a In particular, we showed that linking AuNCs through the ligand holistic description of the nature of the interactions present at the exchange strategy within protein templates can have a strong protein-NC interface. This will allow for the establishment of rules enhancement effect on their linear and nonlinear optical lumi- for predicting optimal protein-NC interaction in order to initiate nescence properties in the NIR-II region . Longer circulation new experiments focused on applications. times and deeper accumulation within the tumor might be Theaim whichshouldberealizedinthe future is theoptimization achieved by post-functionalization strategies, and ligand pro- of the efficiency of protein- NC interactions in the context of tected metal NCs were found to be ideal templates. One of the applications. Present achievements are based on general character- main advantages of ligand protected NCs is the possibility of izations of ligated noble metal nanoclusters representing the class of creating a NC platform with functional moieties appended onto ultrasmall nanomaterials with distinctive structural and optical each surface ligand, dramatically enhancing targeting sensitivity properties, which have yet to be extended beyond the proof of toward cancer materials. principle. This means that the optimal size of NCs and the choice of COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem 3 COMMENT COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 ligandshavetobedesignedfor efficient interaction with proteins in 4. Xie, J., Zheng, Y. & Ying, J. Y. Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 131, 888–889 (2009). the context of improving desirable optical properties, such as high 5. Zare, I. et al. Protein-protected metal nanoclusters as diagnostic and brightness and photo-stability needed for numerous applications in therapeutic platforms for biomedical applications. Materials Today https:// medical diagnostics and medical treatments. The ligands play an doi.org/10.1016/j.mattod.2020.10.027 (2021). additional key role since they participate in drastically enhancing 6. Soleilhac, A., Bertorelle, F. & Antoine, R. Sizing protein-templated gold non-linear optical properties (NLO) of liganded NCs in the NIR nanoclusters by time resolved fluorescence anisotropy decay measurements. Spectrochimica Acta Part A Mol. Biomolecular Spectrosc. 193, 283–288 (2018). regime . Moreover, functionalization can be achieved by ligand 7. Rojas-Cervellera, V., Raich, L., Akola, J. & Rovira, C. The molecular exchange. An example is functionalized aminooxy Au SG NCs 15 13 mechanism of the ligand exchange reaction of an antibody against a (obtained by ligand exchange) interacting with carbonylated proteins. glutathione-coated gold cluster. Nanoscale 9, 3121–3127 (2017). Protein carbonylation at the molecular level on model lysozyme was 8. Bertorelle, F. et al. Tailoring NIR-II photoluminescence of single thiolated investigated by computational chemistry to determine the nature of Au25 nanoclusters by selective binding to proteins. Research Square https:// doi.org/10.21203/rs.3.rs-958149/v1 (2022). bindingbetween theNCs andthe proteincarbonyls (see Fig. 1c). 9. Combes, G. F. et al. Functionalized Au15 nanoclusters as luminescent probes Altogether, this is proof of principle that functionalized liganded for protein carbonylation detection. Commun Chem. 4, 69 (2021). AuNCs can serve for the detection of carbonylation sites and might 10. Bonačić-Koutecký, V. & Antoine, R. Enhanced two-photon absorption of be more efficient than organic dyes. Such rational design of novel ligated silver and gold nanoclusters: theoretical and experimental assessments. functional thiolate-protected metal NCs with controllable surface Nanoscale 11, 12436–12448 (2019). 11. Gunawardene, P. N., Corrigan, J. F. & Workentin, M. S. Golden opportunity: a chemistry could open new avenues towards many other practical clickable azide-functionalized [Au25(SR)18]− nanocluster platform for applications, particularly in bioimaging and early medical diagnostics. interfacial surface modifications. J. Am. Chem. Soc. 141, 11781–11785 (2019). 12. Gran, E. R. et al. Size and ligand effects of gold nanoclusters in alteration of Outlook organellar state and translocation of transcription factors in human primary astrocytes. Nanoscale 13, 3173–3183 (2021). The recent studies mentioned above show both the possibility and 13. Dong, L. et al. Cytotoxicity of BSA-stabilized gold nanoclusters: in vitro and the need to further investigate the nature and the stability of the bio- in vivo study. Small 11, 2571–2581 (2015). NC interface. Advances in electron/X-ray techniques for structural 14. Maysinger, D. et al. Insights into the impact of gold nanoclusters Au10SG10 characterization are absolutely mandatory to better understand the on human microglia. ACS Chem. Neurosci. 13, 464–476 (2022). protein-NC interactions in complex biological media, but also to 15. Colombé, C. et al. Gold nanoclusters as a contrast agent for image-guided surgery of head and neck tumors. Nanomed. Nanotechnol., Biol. Med. 20, 102011 (2019). probe structural and chemical changes that occur in situ and in real 16. Shen, D. et al. Zwitterion functionalized gold nanoclusters for multimodal near time. Mass spectrometry should also bridge the gap to characterizing infrared fluorescence and photoacoustic imaging. APL Mater. 5, 053404 (2017). increasingly complex ligated metal nanoclusters (using zwitterionic 17. Perić, M. et al. Ligand shell size effects on one- and two-photon excitation ligands, proteins, multi-shell ligands) .Advancementsinin situ fluorescence of zwitterion functionalized gold nanoclusters. Phys. Chem. spectroscopic measurements will lead to a better understanding of Chem. Phys. 21, 23916–23921 (2019). 18. Antoine R. Bonacic-Koutecky V. Liganded silver and gold quantum clusters. how protein-NCs perform in biological environments and conditions Towards a new class of nonlinear optical nanomaterials. SpringerBriefs in and will help towards improved designs for their intended applica- Materials. (Springer, Cham, 2018). https://doi.org/10.1007/978-3-319-64743-2_1. tions, including the reinforcement of the link between the AuNC and 19. Comby-Zerbino, C., Dagany, X., Chirot, F., Dugourd, P. & Antoine, R. The the protein for a longer biological stability. emergence of mass spectrometry for characterizing nanomaterials. Atomically Super resolution microscopy with life-time imaging and precise nanoclusters and beyond. Mater. Adv. 2, 4896–4913 (2021). advanced electron microscopy methods allowing for 3D recon- structions from living cells will add to our still limited knowledge of Acknowledgements nanocluster fate in diverse cell types and their physiological and The idea for this manuscript came up during the participation of all four co-authors at pathological conditions. Kinetic parameters showing residence time the Research Workshop “Interdisciplinary Endeavour in Technology at Nanoscale, Water and Environment” (9–11 October 2019), MedILS, Split, Croatia, organized by the of nanoclusters in different organelles are currently not available. head, prof. dr. dr. h. c. Vlasta Bonačić-Koutecký, of the Center of Excellence for Science These would require challenging experimental protocols but they and Technology - Integration of Mediterranean Region, Research, Innovation, Education, could provide much needed information leading to a better idea (STIM-REI) at the University of Split, Croatia. (Contract Number: KK.01.1.1.01.0003, a about organelle-associated proteins and their roles in interacting project funded by the European Union through the European Regional Development with nanoclusters. Human cell organoids are valuable model sys- Fund – the Operational Programme Competitiveness and Cohesion 2014–2020 (KK.01.1.1.01)). The contribution within the activity “Design of novel biosensing tems to explore nanoclusters in a more complex cellular environ- materials and their application in medical diagnostics in biomedicine” extended the ment, complementing those in monolayer cultures and co-cultures. existing theoretical and experimental network (Berlin- Split- Lyon), opening and addi- Computational contributions in establishing rules for designing tional interdisciplinary avenue in medical application in vitro (Montreal) and in vivo appropriate interactions between proteins and nanoclusters can (Grenoble). These research directions resulted in a novel approach towards nanocluster- contribute to motivate new appropriate experiments. The joint bio interaction with proteins. As a chain reaction in science this approach opens new questions to be answered in the future. The co-authors would like to express their efforts of physical and theoretical chemists, material scientists and gratitude to the collaborators: Franck Bertorelle, Martina Perić Bakulić, Željka Sanader biologists are expected to propel protein-protected NCs to ever- Maršić and Evan Rizel Gran. greater achievements in biological applications. These joint efforts will contribute in the future to the further development of new Author contributions nanoclusters with optimized properties. R.A., D.M., L.S. and V.B.-K. contributed equally to the manuscript. Received: 19 February 2022; Accepted: 14 March 2022; Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to Rodolphe Antoine or References Vlasta Bonačić-Koutecký. 1. Kang, X. & Zhu, M. Tailoring the photoluminescence of atomically precise Reprints and permission information is available at http://www.nature.com/reprints nanoclusters. Chem. Soc. Rev. 48, 2422–2457 (2019). 2. Tabarin, T. et al. Absorption enhancement and conformational control of peptides by small silver clusters. Phys. Rev. Lett. 101, 213001 (2008). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 3. Goswami, N., Zheng, K. & Xie, J. Bio-NCs – the marriage of ultrasmall metal published maps and institutional affiliations. nanoclusters with biomolecules. Nanoscale 6, 13328–13347 (2014). 4 COMMUNICATIONS CHEMISTRY | (2022) 5:47 | https://doi.org/10.1038/s42004-022-00665-9 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00665-9 COMMENT Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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