Small Meets Smaller: Effects of Nanomaterials on Microbial Biology

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Small Meets Smaller: Effects of Nanomaterials on Microbial Biology, Pathology, and Ecology Roland H. Stauber,*,† Svenja Siemer,† Sven Becker,† Guo-Bin Ding,†,‡ Sebastian Strieth,† and Shirley K. Knauer*,§ †

Department of Nanobiomedicine/ENT, University Medical Center of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany Institute of Biotechnology, Shanxi University, No. 92 Wucheng Road, 030006 Shanxi, China § Department of Molecular Biology II, Centre for Nanointegration (CENIDE), University Duisburg-Essen, Universitätsstraße 5, 45117 Essen, Germany

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ABSTRACT: As functionalities and levels of complexity in nanomaterials have increased, unprecedented control over microbes has been enabled, as well. In addition to being pathogens and relevant to the human microbiome, microbes are key players for sustainable biotechnology. To overcome current constraints, mechanistic understanding of nanomaterials’ physicochemical characteristics and parameters at the nano−bio interface affecting nanomaterial−microbe crosstalk is required. In this Perspective, we describe key nanomaterial parameters and biological outputs that enable controllable microbe− nanomaterial interactions while minimizing design complexity. We discuss the role of biomolecule coronas, including the problem of nanoantibiotic resistance, and speculate on the effects of nanomaterial−microbe complex formation on the outcomes and fates of microbial pathogens. We close by summarizing our current knowledge and noting areas that require further exploration to overcome current limitations for next-generation practical applications of nanotechnology in medicine and agriculture.

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and prevention of diseases, including cancer or microbial infections.1,5 Whereas our knowledge of the interactions between NMs and human cells has significantly increased over the past decade, surprisingly little is known about the crosstalk of both unintentionally generated or engineered NMs with microorganisms. Although socioeconomically highly relevant, only a minority of microbes are human pathogens. The majority of microbes are biological control elements that are essential for life on earth, including the human body. Such “microsized bioparticles” are exemplified by bacteria, fungal spores, and the huge variety of zoo-/phytoplankton (Figure 1A). Thus, terrestrial and marine ecosystems as well as bioaerosols are exposed to both naturally occurring and anthropogenic NMs. Although interest in these topics is increasing, the majority of studies have focused on the antibacterial activity of engineered NMs rather than comprehensively addressing the diverse aspects of NM−microbe crosstalk for ecosystems and the Anthropocene in general. Notably, all potential NM uptake/exposure routes in humans, animals, and plants are naturally colonized or represent infection paths for diverse microbes (Figure 2).3,4

rofound scientific understanding of rather complex topics has been driven by scientists asking questions such as (1) do nanomaterials’ (NMs) physicochemical properties affect NM−microbe crosstalk? If yes, which properties are most relevant? (2) Microbes come in several different varieties, do NMs interact with all types of microbes? (3) Is NM−microbe complex formation relevant for our desired biotechnological/medical applications? (4) How can we enhance or reduce NM−microbe interactions by targeted chemistry? (5) How do the complexities of (patho)physiological or ecological environments potentially affect NM−microbe crosstalk and practical applications? (6) What is interesting in the future of NM−microbe interactions? In this Perspective, we seek to provide answers to some of these challenges and present strategies and suggestions for how the field can move forward. Do Nanomaterials’ Physicochemical Properties Affect Nanomaterial−Microbe Crosstalk? Nanomaterials are produced naturally, generated unintentionally through anthropogenic sources or engineered for specific applications. The use of engineered NMs in (bio)technology, agriculture, food, personal care products, and medicine is on the rise, meaning that humans, animals, and the environment are increasingly exposed to NMs.1−4 There is also justified hope that NMs may effectively contribute to improving the diagnosis, treatment, © XXXX American Chemical Society

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Figure 1. (A) Relative size range of microbes, cells, and nanomaterials (NMs). (B) Physicochemical characteristics of NMs potentially determine their interactions with and effects on microbes.

Figure 2. (A) Uptake and exposure routes for nanomaterials (NMs) overlap with infection paths for pathogens or are naturally inhabited by microbes. (B) In the environment, microorganisms such as bacteria, microalgae, or fungal spores are exposed to both naturally occurring and engineered NMs.

acknowledged as shaping human development, health, and disease and is increasingly facing NMs.6,7 Nanomaterials’ characteristics define their technological applications, behavior at nano−bio interfaces, and potential toxicology (Figure 1B).4,8 Hence, NMs’ physicochemical “fingerprints” are expected to co-determine their interactions

Although the respiratory tract is a primary target for diseaseinducing microbes and potentially for particulates, the gastrointestinal tract is also an important mediator of interactions with the environment. In addition to enteric pathogens, the complex microbial ecosystems present within the human gastrointestinal tractthe microbiomeis now B

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Table 1. Microbial Surface Structures microbe

surface molecules

size (μm)

shape

ζ-potential

Gram-negative bacteria Gram-positive bacteria fungal spores microphytes/microalgae

phospholipids, liposaccharides peptidoglycan hydrophobins, β-glucan mannan, xylan, alginic acid

0.5−5 0.5−5 2−10 1−500

coccal, rods, spirals, vibrio, filamentous coccal, rods, spirals, vibrio, filamentous sphere, elliptical, tear-shaped, fusiform cylindrical, crescent-shaped, tear-drop, round/oval, rod

negative negative negative negative

and effects on microorganisms (Figure 1B). However, questions remain as to which physicochemical NM characteristics are most relevant and for which types of microbes? Both NMs and microbes come in different shapes and physicochemical varieties;3,5,9 the physical and chemical variations of microorganismal surface structures are important for their pathobiology but may also influence their strengths and types of interactions with nanoparticulates (Table 1). Such variables are exemplified by the molecular compositions of bacterial cell walls, including Gram-positive versus Gram-negative bacteria, or the hydrophobic cell surface of fungal spores.10 The rigid hydrophobin layer of airborne fungal spores makes them resistant to chemical or physical stresses, prevents their effective recognition by the human immune system, and most likely also makes them resistant to the penetration of NM-based antifungals. In addition, microbial shapes, exemplified by coccal versus rod-like bacteria, may also influence NM−microbe interactions (Table 1). Stronger effects of NMs on Gram-positive versus Gram-negative bacteria have been reported, although the shapes of the examined bacteria also varied in these studies.5,9 Hence, the mechanistic details underlying such observations remain to be determined. Notably, in contrast to the tunable surface charge of engineered NMs, most microbes display overall negative surface charge (Table 1). Based on the rules of colloidal electrostatics, it was thus proposed that positively charged NMs might bind more efficiently to microbes than would negatively charged NMs.5,11 However, researchers often modified other NM parameters in addition to NMs’ surface charge in these studies and generally did not investigate in situ complex formation, hampering clear correlations between charge and binding efficiencies.5,11 In contrast, recent studies have demonstrated that various negatively charged NMs were able to bind efficiently to microbes such as bacteria, algae, or fungal spores, displaying overall negative surface charge.3 Moreover, selectively reducing the negative surface charge of NMs by chemical modification but maintaining other parameters did not result in improved binding.3 Notably, synthetic microparticles with similar sizes and surface charges as microbes followed the rules of colloidal electrostatics and did not bind negatively charged NMs. In contrast to synthetic, chemically homogeneous particulates, microbial surfaces may contain exposed, positively charged nanodomains mediating supramolecular contact with negatively charged NMs. Hence, self-assembly of NM−microbe complexes cannot currently be predicted solely by the rules of colloidal electrostatics. In contrast to parameters such as material, shape, or charge, NM size seems to be most critical for NM−microbial binding. Although only a limited selection of NMs was examined, smaller NMs bound more efficiently than larger ones, and microparticles of various materials did not form complexes with bacteria or fungal spores (Figure 3).3 In addition, surface modification with steric molecules, often applied to achieve “stealth” effects for biomedical NMs by reducing overall protein binding and improving biodistribution, also prevents

Figure 3. (A) Nanomaterials’ (NMs) physicochemical properties potentially affect NM−microbe interactions. (B) Size appears to be critical, as smaller silica nanoparticles (NPs; top; ⌀ ∼ 30 nm) bind more efficiently to E. coli bacteria cells than do larger ones (bottom; ⌀ ∼ 140 nm). NP binding visualized by scanning electron microscopy. Note: PEG = polyethylene glycol; PEtOx = poly-2-ethyloxazoline).

binding to bacteria. However, whether these insights will enable the rational engineering of NMs to bind selectively to certain microbes, including more complex bioparticles, such as viruses, algae, or potentially even allergen-relevant pollen, remains to be addressed. However, the impressive progress in the area of graphene two- and three-dimensional engineering deserves serious attention.12 Do Nanomaterials Interact with All Types of Microbes? As microbes come in different varieties, let us imagine a hypothetical aquarium where bacteria, fungal spores, and microalgae are swimming together. Into this hypothetical ecosystem comes an amorphous silica nanoparticle (NP; SiO; diameter 30 nm, negative surface charge): Can we predict to which microsized “bioparticle” the NP would bind first, and why? Although the sizes of these bioparticles, such as bacteria and fungal spores, are comparable and all display an overall negative surface charge, their cell surfaces vary significantly in their physicochemical and molecular compositions. As displayed in Figure 4, SiO NPs associated with the bioparticles in the following order: fungal spores > microalgae > bacteria. These observations indicate that although NMs seem to interact with all type of microbes, their affinity for microbial surfaces differs significantly. At the moment, we do not know the supramolecular determinants mediating high- versus lowaffinity adsorption. However, such knowledge would potentially enable the design of NMs that bind selectively to certain microbes but not to others. Moreover, we could perhaps exploit such NMs as sensors to probe the structure and chemistry of microbes as well as dynamic changes during their life cycle. Is Nanomaterial−Microbe Complex Formation Relevant for the Desired Biotechnological/Medical Applications? The lack of novel antibiotics combined with the increasing occurrence of multi-drug-resistant bacteria repreC

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although the resultant effects on NM−pathogen complex formation were not investigated.19 We therefore strongly advocate for researchers to discriminate between effects mediated by direct versus indirect NM−microorganism crosstalk in order to dissect the mechanisms of complex formation and ultimately to contribute to a more complete picture than that which has been painted by the various and sometimes contradictory studies that have been reported. Additional biotechnological applications for which controllable, tuned interactions between NMs and microbes would be relevant include water and solid waste purification and bioenergy production, among others. We challenge the field to investigate the effects of such crosstalk on natural aquatic or terrestrial ecosystems, although such complex systems are certainly challenging to study and require tiered analytical approaches in order to draw mechanistic insights and meaningful conclusions. Chemical Strategies To Control Nanomaterial− Microbe Interactions. Because NM−microbe interactions are expected to be influenced by multiple factorsranging from differences due to the varying types of NMs and microbes to environmental conditions, such as temperature and ion or biomolecule concentrationsreliable and complementary analytical methods are required to obtain mechanistic insights. Powerful techniques in the areas of materials sciences and microbiology now make it possible to investigate NM− microbe crosstalk with unprecedented throughput and resolution. However, most studies have not applied a tiered experimental pipeline, which would enable a comprehensive characterization of details and scientific questions from analytical to in situ, in vitro, in vivo, and, ultimately, in silico.

Figure 4. Nanoparticles (NPs) adsorb to various microbes and human cells with different kinetics. In situ complex formation of red or green fluorescent silica NPs with autofluorescent Aspergillus fumigatus spores (green), autofluorescent microalgae (magenta), autofluorescent E. coli (red). Indicated living microbes or cells were incubated with NPs for various time points, washed, and analyzed by microscopy. Earliest time points after which NP adsorption was detectable are indicated.

sents one of the major health challenges worldwide.9,11 Nanomaterials-based approaches may be able to present reinforcements of common antimicrobial strategies. Thus, NMs are being intensively investigated as smart tools for the diagnosis of pathogens,13 as more efficient antibiotic drug carriers,14 or, based on the antimicrobial effects of certain NMs, as novel antibiotics.11,15 The development of antibacterial nanotherapeutics has mainly focused on metal, metal oxide, semiconductor, or polymer NMs acting via different mechanisms.11,15,16

Because nanomaterial−microbe interactions are expected to be influenced by multiple factors, reliable and complementary analytical methods are required to obtain mechanistic insights.

Although nanomaterials seem to interact with all type of microbes, their affinity for microbial surfaces differs significantly. Pathophysiological environments for practical clinical applications, such as the circulatory system, the lungs, wounds, or the oro-gastrointestinal tract, are highly dynamic, and thus, NMs will directly encounter pathogens for only a short time. Independent of their antimicrobial mode of action, nano-based antibiotics should physically bind to the pathogens’ cell surfaces to be most effective, in both pathophysiological and agriculturally relevant environments.3 Recent data demonstrate that complex formation is required for NMs’ efficient bactericidal activity, although the relevance of NM adsorption as potential antifungals has not yet been fully studied.3 The majority of previous studies investigating antibiotic NMs or the impact of NMs on the microbiome, however, did not apply methods to discriminate between effects mediated directly by NM−microbiota complex formation or indirectly by NM dissolution versus secondary effects by released ions.17,18 The well-known toxicity of certain metal ions, such as silver ions, being released from micro- or nanosize metals or from dissolved metal salts should not be classified as nanotechnology-driven discovery. Problems of NM resistance and mechanistic strategies to overcome these limitations for next-generation practical applications remain unresolved challenges. In addition to known genetic resistance, recent reports also suggest NM-specific resistance mechanisms,

A critical initial step in this pipeline is to investigate if and under what conditions NM−microbiota complex formation occurs in situ in relevant environmental scenarios. Often, NM− microbe interactions are analyzed by electron microscopy (EM) only. Despite advantages concerning resolution and visualization of structural details, most EM techniques are low throughput and require harsh fixation and staining procedures, including chemical cross-linking, drying, and high vacuum. Such procedures can result in artifacts, such as membrane rupture of bacteria or fungal spores, leading to the impression that NMs can easily penetrate the surface of microbes. An efficient method to study exposure and contact scenarios occurring in realistic dynamic physiological and ecologically relevant environments under controllable conditions in situ is depicted in Figure 5. Here, NMs and microbes are coincubated for various time periods. Subsequently, NM− microbiota complexes can be harvested by mild centrifugation, whereas unbound NMs remain in the supernatant and are thereby removed. Additional variable washing steps, such as using high levels of salt, high temperatures, or detergents, can be included to probe the strength and chemistry of NM binding further. Isolated NM−microbe complexes can subsequently be used for further experimental analyses using D

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most Gram-negative bacteria produce pili for their adhesion to (bio)material surfaces and/or cell membranes. These pili contain bacterial adhesins that interact with carbohydrates. Thus, targeting these receptors with synthetic glycosylated structures enables us to modulate bacterial adhesion, potentially also binding to NMs.22−25 Moreover, electrostatic interactions represent a classical approach for supramolecular interactions. The overall negative bacterial surface charge which is primarily due to the dense glycocalyx, which contains numerous deprotonated carbohydratesenables the binding of cationic supramolecular decoys. It is surprising, however, that even negatively charged NMs efficiently bind to bacteria, indicating that the release of the NMs’ high surface energy overrides electrostatic repulsion. In contrast, the selective detection of Gram-positive bacteria by supramolecular self-assembled agents is challenging due to the lack of a second outer membrane or highly specific carbohydrates or receptors (Figure 6). However, lipoteichoic acid, which consists of cross-linked carbohydrates, is one key structure that is addressable by electrostatic interactions. Membrane-stabilizing bacterial biosurfactants, such as surfactin, are another potential target; biosurfactants act as protective agents against other bacterial competitors, which can be targeted by diacetylenic lipids, for example.26 Although innovative strategies and targets for supramolecular interactions on the surface of bacterial pathogens have been established, we are awaiting experimental verification of whether the integration of such structures onto nanosized objects will enable us to control microbe recognition and NM−microbe complex formation (Figure 6). Notably, targeting the surface of fungal pathogens by such (or alternative) strategies has been neglected to date. How Do the Complexities of (Patho)physiological or Ecological Environments Potentially Affect Nanomaterial−Microbe Crosstalk? Supramolecular chemistry and NM synthesis work well in the “test tube”; however, one may ask, “What happens in real life?” Various biomolecules can rapidly adsorb onto all known NMs when entering complex physiological or ecological environments. The properties and fates of such “coronated” NMs in these environments often differ significantly from those of pristine, manufactured NMs.4,8,27 Physiological environments such as plasma, saliva, intestinal fluids, or lung surfactant (lipoprotein complexes) contain proteins, lipids, and polysaccharides; these environments are complex but rather predictable in their composition. In contrast, natural aquatic or terrestrial environments with their multitude of biotic and abiotic matter represent additional complexity. Hence, analytical and functional

Figure 5. Workflow to investigate parameters of nanomaterial (NM)−microbe complex formation in situ. (A) Following coincubation in distinct media under controlled conditions, NM− microbe complexes are harvested by mild centrifugation. Unattached NMs remain in the supernatant and are removed. (B) NM−microbe complexes can subsequently be analyzed via different methods, such as fluorescence microscopy.3

in vitro and in vivo models. By employing fluorescent NMs in combination with genetically modified microbes to produce various autofluorescent proteins, NM−microbe interactions can be monitored and even be quantified by microscopy-based high-throughput/high-content imaging systems.3 Once NM−microbe interactions are confirmed, targeted (supramolecular) chemistry may be exploited as a strategy to enhance or to reduce NM−microbe complex formation. However, because such strategies need to consider supramolecular principles concerning microbial surfaces, researchers are aiming at specific targets on the surface of bacterial and fungal pathogens to fine-tune and/or inhibit their pathobiology. Bacterial pathogens are divided into two main classes: Gram-positive and Gram-negative. Their distinct surface chemistries not only affect NM−bacteria interactions but also the success of targeted supramolecular chemistry in general (Figure 6). Gram-negative bacteria consist of two membranes with integrated porins and membrane proteins, which can be problematic to address with supramolecular platforms. The main targets are presented on the outer membrane surface. The glycocalyx, which consists of liposaccharides, is a primary target for specific interactions with carbohydrate-recognizing structures, such as cationic amphiphiles.20,21 Furthermore,

Figure 6. Nanomaterial (NM) binding depends on microbial surface structures and nanoparticle size. (A) Gram-negative bacteria. (B) Grampositive bacteria. (C) Hypothetical nanotools for the specific targeting of bacteria. Note: Objects are not drawn to scale. E

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Figure 7. Effects of nanomaterial (NM) biomolecule coronas on NM−microbe interactions. NM−bacteria complex formation is reduced by the formation of a biomolecule corona on the NM. In the presence of biomolecules, NMs acquire a biomolecule corona, reducing NM− microbe interactions to various extents. Fluorescence microscopy demonstrates in situ complex formation of autofluorescent green bacteria with red fluorescent silica nanoparticles (NPs).

Figure 8. “Smart” nanomaterials (NMs) or agents enhancing NM−bacteria complex formation in wound infections are expected to show improved antibacterial and wound-healing activity. Note: NP = nanoparticle.

investigations of such natural “eco-coronas” have just begun.4 Moreover, heterogeneous, mixed coronas may form when NMs are released into ecosystems and/or subsequently enter other environments, such as animals or humans. Relationships between NMs’ characteristics and the nature of the corona is far from trivial and currently cannot be predicted under realistic conditions. Nevertheless, it is the corona of macromolecules that is thought primarily to interact with surrounding systems and, thereby, constitutes a major element of NMs’ desired activities as well as their pathobiological and ecological identities. Hence, it is surprising that biomolecule coronas’ effects on NM−microbe crosstalk have only recently been studied. The majority of studies report effects of various antibacterial NMs in the absence of macromolecules and do not investigate corona formation.5 In a recent study, Westmeier et al. systematically investigated the effects of environmental factors, including corona-forming biomolecules, on NM−bacteria interactions.3 The researchers reduced NM−microbe complex formation by the assembly of various biomolecule coronas, including plasma proteins, lungsurfactant lipids, and environmental humic acids (Figure 7). Because bacteria that were preincubated with biomolecules were still able to bind NMs, adsorption of biomolecules to

NMs and not to microbes interfered with complex formation. High biomolecule concentrations completely prevented NM binding to bacteria. This study suggests that the extent of NM−microbe interactions, in general, is influenced by biomolecule coronas assembling at first contact with NMs and microbes. Which types of biomolecules, such as proteins, lipids, or complex organic molecules, are most effective in mediating these effects and for which types of microbes these effects are most relevant remain to be determined. Panácě k and colleagues proposed that the bacterial protein flagellin triggers resistance to silver NPs by an unknown mechanism.19 Because biomolecules produced by bacteria, including flagellin, also form coronas on antimicrobial NMs, the study independently confirmed that NM coronation seems to be the underlying mechanism leading to bacterial NM resistance by reducing NM−bacteria complex formation.3 No resistance developed under corona-free conditions and bacterial resistance could be induced by different proteins or lipids, independent of their biological function.3,19 Such physical shielding may be one reason why the antibiotic activity of NMs is often significantly impaired under pathophysiologically relevant conditions. Hence, it may be useful to investigate if the nanoformulations presented in various studies as novel antibiotics are indeed still active under F

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not only for basic or applied nanoscience but also for understanding the impact of bioparticles and bioaerosols on the natural world and on human health. Although relationships between NMs’ physicochemical characteristics and microorganisms have just begun to be investigated in detail, some general principles have emerged (Table 2). In general, NMs appear to be capable of interacting spontaneously with various types of microbes, although to different degrees, the specifics of which cannot yet be reliably predicted. In addition, whether the often laborious implementation of additional chemical “recognition elements” as building blocks is needed to drive NM−microbe complex formation for a desired application (and, if so, to what extent) needs to be fully resolved or reinvestigated.5 In this Perspective, we further discussed how stealth modification strategies enable us to reduce NM− bacteria interactions and described the relevance of biomolecules for practical applications. Numerous challenges remain, which will require collaboration between specialized and sometimes still-isolated disciplines to investigate this crosstalk and to elucidate its ecological and pathobiological relevance. In addition to the tuning of antibiotic NMs, even nonbactericidal NMs, which are already part of consumer products, might influence the pathobiology of bacteria through the formation of NP− bacteria hybrid structures: the microbial infection cycle, NM− pathogen uptake into target cells, and how the human immune systems “sees” such NM−pathogen hybrids all seem to be influenced.3 Epithelial target and/or immune cells primarily face NP−microbe hybrid structures rather than pristine microbial surfaces (Figure 9). Thus, cellular receptors may respond quantitatively and/or qualitatively differently to such hybrid structures, ultimately modulating downstream signaling pathways that are important for cellular and organismal homeostasis, clearance of pathogens, and/or disease. Given the relevance of the gut microbiome, one may also ask if the increasing use of nanosized food additives potentially represents a selective pressure on the gut microbiome or if this use could be exploited to achieve positive, preventive “side effects” by inhibiting enteric pathogens, such as gastric carcinoma-associated Helicobacter pylori.3,7 Collectively, the broad ecological and pathobiological relevance of these emerging insights for microorganisms, in general, remains to be determined. Such knowledge will also

realistic pathophysiological conditions in clinical scenarios, such as in wounds, the gastrointestinal tract, or the blood system. We speculate that physicochemical strategies enhancing NM−pathogen interactions under pathophysiologically relevant conditions will significantly increase the antibiotic activity of nano-based agents, not only against bacteria but also against microbes in general, including fungal spores. As illustrated in Figure 8, formulations containing either “smart” NMs or agents triggering NM−pathogen complex formation in wound infections are expected to be of great interest for broad clinical applications. Furthermore, such strategies may also be exploited to modulate bacteria−NM assembly to improve various biotechnological, agricultural, or biomedical applications. We are still awaiting the discovery of such “smart” nanotools by innovative (supramolecular) chemistry though, as previous strategies to inhibit corona formation by “stealth” molecules unfortunately also reduced NM−pathogen complex formation.3

OUTLOOK, CHALLENGES, AND FUTURE DIRECTIONS The complex interplay of NMs with microbes is an emerging hot topic: ‘When small meets smaller’ is of growing relevance, Table 2. Parameters Suggested To Affect Nanomaterial (NM)−Microbe Complex Formation parameter NM characteristic size charge

influence highly relevant; improved binding for smaller NMs; no binding for microparticles (⌀ > 500 nm) independent (efficient binding of negatively or positively charged NMs) independent (binding of various NMs) important (prevents NM−bacteria interaction)

material “stealth” surface functionalization physiological/ecological parameter biomolecules concentration-dependent reduction of NM−microbe interaction exposure time rapid adsorption, though kinetics dependent on microbes’ surface structure and chemistry temperature no effect on complex formation (4−55 °C)

Figure 9. Model illustrating how nanomaterial (NM)−microbe assembly may affect pathobiology. Naturally occurring or engineered NMs can adsorb to bacterial surfaces, interacting with structures such as liposaccharides, adhesins, or lipoteichoic acid (LAA). Mechanistically, important components of the innate immune response, including Toll-like receptors (TLRs) and cytokines might be involved. Macrophages may recognize silica nanoparticle (SiNP)−bacterial surface hybrids as novel pathogen-associated molecular patterns via TLR2/4 and/or the class B scavenger receptor SR-B1.28 Co-stimulation of TLR2/4 and SR-B1 receptors may alter cytokine production and overall immune responses. Simultaneous binding of NP−microbe hybrids to TLRs as well as to the SR-B1 receptor may influence phagocytosis and subsequent clearance of NM−microbe complexes. G

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Figure 10. Schematic illustration of the strategic plan to understand the mechanisms and relevance of NM−microbe crosstalk, employing a tiered experimental pipeline from chemical design to analytical, in situ, in vitro, and in vivo investigations. Supramolecular design and controlled synthesis of “smart” nanotools enable us to analyze their interactions with different types of microbes via various technologies, including analytical electron and in situ high-throughput microscopy. Effects and mechanisms can subsequently be investigated using twodimensional (2D) cell monocultures as well as coculture systems to study the direct interaction and uptake of complexes, as well as effects on cellular communication via soluble messengers, such as cytokines. Organoids mimic a miniaturized and simplified version of an organ produced in vitro in three dimensions, relevant to study processes at the nano−bio interface. Organ-on-a-chip integrates advanced threedimensional (3D) tissue engineered constructs with microfluidic network systems and dynamic communication between cell types, microbes as well as nanomaterial (NM)-covered microbiota can be controlled. Galleria mellonella is suggested as a simple and cheap model for studying microbial pathogens and/or the testing of antimicrobial NMs. Mouse models enable us to investigate biodistribution, toxicity, pathogenicity, and immune responses using gnotobiotic, specific-pathogen-free, or conventional mice. Ultimately, stringently controlled human studies should only be performed upon completion of the experimental pipeline, proven safety, and efficacy. Note: HTS = highthroughput screening; NP = nanoparticle.

foster the “safe by design” concept of applied nanotechnology, which is increasingly requested by many regulatory networks. Our toolbox of sophisticated techniques and models available to study nanotechnology and microbiology needs to be combined in tiered experimental pipelines (Figure 10). Moreover, cross-disciplinary communication in academia and industry is needed to close existing knowledge gaps. We further emphasize the need to discriminate between effects mediated by direct versus indirect NM−microorganism crosstalk to dissect underlying mechanisms and ultimately contribute to a more complete picture than has been painted by observations to date. We further call for comprehensive studies investigating the effects of NM−microorganism complex formation on the environment and human health, including bacterial and airborne fungal infections, the microbiome, and allergies induced by bioparticles such as pollen. Such knowledge is needed not only to understand and to minimize potential “nanotoxicity” but also to expand NMs for improved future applications and to shape and to control (pathological) microorganisms in biotechnology, agriculture, and translational biomedicine.

STR_STA_1014, NanoTransMed which is co-founded by the European Regional Development Fund (ERDF) in the framework of the INTERREG V Upper Rhine program, the Swiss Confederation and the swiss cantons of Aargau, BaselLandschaft and Basel-Stadt.

ABBREVIATIONS NMs, nanomaterials; NP, nanoparticles; SEM, scanning electron microscopy; SiO, silica NPs blue; SiO-G, silica NPs green; SiO-R, silica NPs red. REFERENCES (1) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20−37. (2) Peterson, E. C.; Ewing, L. E. Nanomedicine: Going Small To Beat the High. Nat. Nanotechnol. 2016, 11, 580−581. (3) Westmeier, D.; Posselt, G.; Hahlbrock, A.; Bartfeld, S.; Vallet, C.; Abfalter, C.; Docter, D.; Knauer, S. K.; Wessler, S.; Stauber, R. H. Nanoparticle Binding Attenuates the Pathobiology of Gastric CancerAssociated. Nanoscale 2018, 10, 1453−1463. (4) Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H. The Nanoparticle Biomolecule Corona: Lessons Learned - Challenge Accepted? Chem. Soc. Rev. 2015, 44, 6094−6121. (5) Gupta, A.; Landis, R. F.; Rotello, V. M. Nanoparticle-Based Antimicrobials: Surface Functionality is Critical. F1000Research 2016, 5, 364. (6) Sonnenburg, J. L.; Backhed, F. Diet-Microbiota Interactions as Moderators of Human Metabolism. Nature 2016, 535, 56−64. (7) McClements, D. J.; Xiao, H.; Demokritou, P. Physicochemical and Colloidal Aspects of Food Matrix Effects on Gastrointestinal Fate of Ingested Inorganic Nanoparticles. Adv. Colloid Interface Sci. 2017, 246, 165−180. (8) Feliu, N.; Docter, D.; Heine, M.; Del Pino, P.; Ashraf, S.; Kolosnjaj-Tabi, J.; Macchiarini, P.; Nielsen, P.; Alloyeau, D.; Gazeau, F.; Stauber, R. H.; Parak, W. J. In Vivo Degeneration and the Fate of Inorganic Nanoparticles. Chem. Soc. Rev. 2016, 45, 2440−2457.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Roland H. Stauber: 0000-0002-1341-4523 Guo-Bin Ding: 0000-0002-3012-179X Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Grant support was provided by Impulsfonds Rheinland-Pfalz/ BioMAS, NMFZ, InnerunivFFI, Biomatics, DFG_SFB1093/ H

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DOI: 10.1021/acsnano.8b03241 ACS Nano XXXX, XXX, XXX−XXX